Thrombospondin-1 type 1 repeat polypeptides

ABSTRACT

Thrombospondin-1 (TSP-1) is a potent inhibitor of tumor growth and angiogenesis. To elucidate the molecular mechanisms that are involved in the inhibition of tumor growth by the type 1 repeats (TSRs), recombinant versions of these motifs have been produced and have been assayed for their ability to inhibit the growth of experimental B16F10 and Lewis lung carcinomas. Recombinant proteins that contain all three TSRs (3TSR) or the second TSR with (TSR2+RFK) or without (TSR2) the transforming growth factor beta (TGFβ) activating sequence (RFK) have been expressed in Drosophila S2 cells. A recombinant protein containing all three type 1 repeats of TSP-2 and a recombinant protein containing the second TSR with the RFK sequence altered to QFK have also been produced. The data indicate that the TSRs inhibit tumor growth by inhibition of angiogenesis and regulation of tumor cell growth and apoptosis. The regulation of tumor cell growth and apoptosis is RFK-dependent while the inhibition of angiogenesis is not. The invention relates to polypeptides based on the amino acid sequence of human TSP-1 type 1 repeats. The polypeptides, variants, fragments and mutants thereof can be made by recombinant methods or can be made by chemical synthesis. The polypeptides can be formulated into pharmaceutical compositions and used in methods of therapy to reduce tumor growth.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 60/207,994 filed May 26, 2000, the entire teachings of which are incorporated herein by reference.

GOVERNMENT SUPPORT

[0002] The invention was supported, in whole or in part, by a grant HL28749 from the National Heart, Lung and Blood Institute of the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Thrombospondin-1 is a potent inhibitor of angiogenesis (for a review, see Dawson, D. W. and Bouck, N. P., “Thrombospondin as an inhibitor of angiogenesis,” In: B. A. Teicher (eds.), Antiangiogenic Agents in Cancer Therapy, pp. 185-203, Totowa, N.J.; Humana Press, Inc., 1999). It inhibits endothelial cell growth, migration and tube formation in vitro (Panetti, T. S., et al., J. Lab. Clin. Med., 129: 208-216, 1997). In vitro assays have shown that platelet thrombospondin-1 is involved in thrombosis, fibrinolysis, wound healing, inflammation, tumor cell metastasis and angiogenesis. The major form of thrombospondin secreted by platelets and endothelial cells is TSP-1. Thrombospondin-1 (TSP-1) is an angiogenesis inhibitor. Thrombospondin-1 has three copies of the TSR. TSP-1 is a trimeric molecule. Thus, the fully assembled protein contains nine TSRs.

[0004] The ingrowth of new capillary networks into developing tumors is essential for the progression of cancer. As pointed out in a review by Folkman (Folkman, J., Proc. Natl. Acad. Sci. USA 95: 9064-9066, 1998), antiangiogenic therapy has little toxicity, does not require the therapeutic agent to enter tumor cells or cross the blood-brain barrier, controls tumor growth independently of growth of tumor cell heterogeneity, and does not induce drug resistance. Thus, the development of pharmaceuticals that inhibit the process of angiogenesis or can inhibit abnormal cell proliferation by other mechanisms is an important therapeutic goal.

SUMMARY OF THE INVENTION

[0005] The invention involves the use of a recombinant or synthetic version or a segment of the type 1 repeat domain (TSR) of human thrombospondin-1 (TSP-1) or variants, fragments or mutants thereof to inhibit tumor growth by angiogenic or other activity. In particular, good anti-tumor activity is obtained if the protein or protein fragment employed includes the KRFK sequence (SEQ ID NO: 28) from the C-terminal end of the first TSR and a sufficient portion of the N-terminal end of the second TSR (through the first disulfide bond) to give the protein or protein fragment tertiary structure similar to the wild-type TSRs. A cDNA encoding amino acids 411 through 473 of human TSP-1 (SEQ ID NO: 22) has been cloned into the pMT/BiP/V5His A expression vector. The recombinant protein has been expressed and purified by standard protocols. This protein is a potent inhibitor of tumor growth in mice. The growth of B16F10 melanoma cells and Lewis lung carcinoma cells is inhibited by 86% and 77%, respectively, with a single daily interperitonial dose of 2.5 mg/kg/day. The inclusion of the sequence KRFK (SEQ ID NO: 28) at the N-terminus of the recombinant protein significantly increased the anti-tumor activity of this protein. The protein that contains the KRFK sequence (SEQ ID NO: 28) is termed broadly herein a polypeptide comprising the second type 1 repeat of human TSP-1, but not comprising the RFK sequence, or the other domains of TSP-1; also, a “TSR2+RFK polypeptide or protein.” At 1 mg/kg/day the TSR2+RFK protein described in the Examples inhibited B16F10 tumor growth by 80%. The protein that lacks the KRFK sequence is termed broadly herein a polypeptide comprising the second type 1 repeat of human TSP-1, and the RFK sequence, but not the other domains of TSP-1; also, a “TSR2 polypeptide or protein” The TSR2 protein described in the Examples inhibited tumor growth by 38%. This result is contrary to previously published results using synthetic peptides and an orthotopic model of breast cancer, in that the KRFK sequence (SEQ ID NO: 28) was previously reported to lack anti-tumor activity.

[0006] The mechanism of action for the inhibition of tumor growth by a TSR2+RFK protein involves inhibition of angiogenesis, induction of tumor cell apoptosis and inhibition of tumor cell proliferation. The latter two activities appear to require the KRFK sequence (SEQ ID NO: 28) in that these effects are not observed with the TSR2 protein. The KRFK sequence (SEQ ID NO: 28) of TSP-1 has not been reported to induce tumor cell apoptosis previously.

[0007] The invention relates to polypeptides based on the amino acid sequence of human thrombospondin 1, and more specifically relates to: (1) a polypeptide comprising the three type 1 repeats of human TSP-1, but not comprising the other domains of TSP-1 (a 3TSR or 3TSR (TSP1) polypeptide or protein); (2) a polypeptide comprising the second type 1 repeat of human TSP-1, but not comprising the RFK sequence or the other domains of TSP-1 (a TSR2 polypeptide or protein); (3) a polypeptide comprising the second type 1 repeat of human TSP-1 and the RFK sequence, but not the other domains of TSP-1 (a TSR2+RFK polypeptide or protein); (4) a polypeptide comprising the second type 1 repeat of human TSP-1 and the RFK sequence altered to QFK, but not the other domains of TSP-1 (a TSR2+QFK polypeptide or protein), and (5) a polypeptide comprising all three type 1 repeats of human TSP-2, but not the other domains of TSP-2 [a 3TSR (TSP2) polypeptide or protein]. See, for the domains of TSP-1, Table 1.

[0008] Further embodiments are fragments, variants and mutants of these polypeptides having amino acid sequences that differ from the type 1 repeat segment of the human TSP-1 or human TSP-2. Variants can have, for example, flanking amino acid sequence, amino acid substitutions, deletions, or additions, or some combination. The polypeptides, variants, fragments and mutants can be made by recombinant methods or can be made by chemical synthesis, using L-amino acids, analogues or derivatives thereof, or D-amino acids or related compounds, or they can be made of only L-amino acids.

[0009] Also described herein are nucleic acid molecules involved in the making of constructs encoding the polypeptides described herein, vectors, and host cells that can be used in recombinant methods of producing the polypeptides.

[0010] The polypeptides can be formulated into pharmaceutical compositions and used in methods of therapy to reduce tumor growth. For example, a TSR2+RFK polypeptide, or a conservative variant thereof, can be combined with a pharmaceutically acceptable carrier. A fragment of the TSR2+RFK with up to 30 consecutive amino acid residues deleted relative to the TSR2+RFK polypeptide having SEQ ID NO: 22 can also be formulated into a pharmaceutical composition. Further, variants of the TSR2+RFK polypeptide, especially conservative variants, and variants at least 70%, 80%, 90% or 95% identical in amino acid sequence to TSR2+RFK can be formulated with a pharmaceutically acceptable carrier.

[0011] The TSR2+RFK polypeptides, fragments, variants and mutants thereof, as well as other polypeptides described herein, can be administered to a human patient in an amount sufficient to induce apoptosis, inhibit neovascularization, or otherwise inhibit growth in cancerous cells (e.g., cells within a tumor).

[0012] A further embodiment of the invention is a method of killing cancerous cells in a patient, comprising administering to said patient an amount of a TSR2+RFK polypeptide fragment sufficient to induce apoptosis in said cancerous cells, wherein said fragment has at least about the same apoptotic and anti-angiogenic activity as TSR2+RFK as illustrated in the Examples, and has up to 30 consecutive amino acids deleted from the carboxy terminus of TSR2+RFK. In variations of the method, a variant which is at least 70%, 80%, 90% or 95% identical in amino acid sequence to TSR2+RFK is administered to a patient, wherein the variant can be combined with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a bar graph showing TGFβ activation by the TSR2+RFK protein of the Examples. Saline, the TSR2 or the TSR2+RFK recombinant proteins (5.0 μg/ml) were incubated with B16F10 cells overnight in serum-free DMEM. The levels of active and total TGFβ were determined using the MLEC assay (Abe, M., et al., Anal. Biochem, 216:276-84, 1994). The asterisk indicates p<0.005 relative to control.

[0014]FIGS. 2A and 2B are graphs showing the effect of TSP-1 and the TSR-containing recombinant proteins of the Examples on endothelial cell migration. HDMEC migration in the presence of varying concentrations of TSP-1 (FIG. 2A, open squares), 3TSR (FIG. 2A, filled diamonds), TSR2 (FIG. 2B, open squares) or TSR2+RFK (FIG. 2B, filled diamonds) was determined after four hours. Migration in response to FGF-2 in the absence of inhibitors was considered 100% migration and migration in the absence of FGF-2 was considered 0% migration.

[0015]FIGS. 3A and 3B are graphs showing growth of B16F10 tumors in mice treated with platelet TSP-1 (FIG. 3A) or 3TSR protein (FIG. 3B). Each treatment group contained six mice. Treatment was initiated four days after subcutaneous injection of 1×10⁶ tumor cells. The mice received one IP injection each day of saline (open squares), or 0.25 mg/kg/day (open circles), 1.0 mg/kg/day (filled triangles) or 2.5 mg/kg/day (filled circles) of 3TSR protein as described in the Examples. Tumor volume was determined every other day with a dial caliper. The mean and standard error are plotted.

[0016]FIG. 4 is a graph showing growth of Lewis lung carcinoma in mice treated with recombinant proteins. Each treatment group contained six mice. Treatment was initiated four days after subcutaneous injections of 1×10⁶ tumor cells. The mice received an IP injection each day of saline (filled squares), or the 3TSR protein (filled circles) or the TSR2+RFK protein (filled triangles). The recombinant proteins were used at a dose of 2.5 mg/kg/day. Tumor volume was determined every other day with dial caliper. The mean and standard error are plotted.

[0017]FIGS. 5A and 5B are graphs showing the growth of B16F10 tumors in mice treated with TSR2+RFK protein (FIG. 5A) or TSR2 protein (FIG. 5B). Each treatment group contained six mice. Treatment was initiated four days after subcutaneous injection of 1×10⁶ tumor cells. The mice received one IP injection each day of saline (open squares), or 0.25 mg/kg/day (open circles), 1.0 mg/kg/day (filled triangles) or 2.5 mg/kg/day (filled circles) of recombinant protein. Tumor volume was determined every other day with a dial caliper. The mean and standard error are plotted.

[0018]FIGS. 6A, 6B and 6C are bar graphs showing the quantitation of blood vessels (FIG. 6A), tumor cell apoptosis (FIG. 6B), and tumor cell proliferation (FIG. 6C). Tumors from mice treated with saline (solid bars), 1.0 mg/kg/day of TSR2+RFK protein (hatched bars) or 1.0 mg/kg/day of TSR2 protein (shaded bars) were fixed and tissue was prepared for histology. The asterisk indicates p<0.005 as compared to control in all cases except the level of apoptosis induced by TSR2 (FIG. 6B, shaded bar) where p≦0.025. A comparison of the vessel density for the mice treated with the TSR2+RFK (FIG. 6A, hatched bar) or TSR2 (FIG. 6A, shaded bar) yields p≦0.05.

[0019]FIG. 7 is a representation of the amino acid sequence of human TSP-1 (SEQ ID NO: 1). The type 1 repeats of TSP-1 are, as illustrated here, 1) amino acids 361-416; 2) amino acids 417-473; and 3) amino acids 474-530. Also shown in FIG. 7 are the complete type 2 repeats, the type 3 repeats, and the procollagen homology region.

[0020]FIG. 8 is a bar graph showing the effect of treatment with recombinant, TSP-1 peptides or controls on the tumor volume from melanoma B16F10 in C57BL/6 mice.

[0021]FIG. 9 is a bar graph showing the effect of treatment with recombinant TSP peptides or saline as control on the tumor volume from melanoma B16F10 in C57BL/6 mice.

[0022]FIG. 10 is a representation of the amino acid sequence of mouse thrombospondin 2, including signal sequence (SEQ ID NO: 24). See National Center for Biotechnology Information Accession No. NP 035711, PID g6755779, as in Laherty, C. D. et al., J. Biol. Chem. 267(5):3274-3281, 1992.

[0023]FIG. 11 is a representation of the amino acid sequence of human thrombospondin 2, including signal sequence (SEQ ID NO: 26). See National Center for Biotechnology Information Accession No. TSHUP2, PID g1070641, as in LaBell, T. L. et al., Genomics 12(3):421-429, 1992.

DETAILED DESCRIPTION OF THE INVENTION

[0024] A description of preferred embodiments of the invention follows.

[0025] The data presented here show that administration of recombinant proteins (also herein: “polypeptides”) that contain the TSRs of TSP-1 or the TSRs of TSP-2 inhibit the growth of experimental tumors. Inhibition has been observed with both B16F10 melanoma and Lewis lung carcinoma cell lines. The 3TSR and TSR2+RFK proteins are relatively potent, showing 70%-80% inhibition of tumor volume at 2.5 mg/kg/day. This concentration is 100-300 nmoles/kg/day. Injection with intact TSP-1 protein at 0.25 mg/kg/day (0.6 nmoles/kg/day) resulted in a 56% reduction of tumor growth, indicating that the intact protein is considerably more active than the recombinant proteins at low doses.

[0026] Several activities of the TSRs may contribute to the inhibition of tumor growth. These include the ability to (1) inhibit angiogenesis, (2) reduce tumor cell proliferation, (3) activate TGFβ, and (4) regulate extracellular proteases. The results described here show that systemic treatment with the TSRs of TSP-1 or TSP-2 reduces vessel density in tumors.

[0027] The rate of tumor cell apoptosis is a key factor in the determination of the rate of tumor growth (Parangi, P., et al., Proc. Natl. Acad. Sci. (USA), 93: 2002-2007, 1996; Naik, P. N., et al., Genes & Dev., 10:2105-2116, 1996). Described herein are results showing that TSP-1 proteins that include the RFK sequence significantly increase the rate of tumor cell apoptosis. Whereas TSP-1 has been reported to inhibit tumor cell proliferation and induce endothelial cell apoptosis, this study demonstrates that the TSR-containing proteins induce tumor cell apoptosis (Guo, NH., et al., Cancer Res., 57:1735-1742, 1997; Guo, N.-H., et al., Cancer Res., 58: 3154-3162, 1998). This activity is enhanced by the addition of the DKRFK (SEQ ID NO: 2) sequence at the NH₂-terminus, because the TSR2 protein did not increase the level of apoptosis of the tumor cells to the same extent as the TSR2+RFK protein. An increase in tumor cell apoptosis is frequently associated with anti-angiogenic therapy due to the decrease in nutrients that is associated with the decrease in blood supply. These data herein indicate that increased tumor cell apoptosis occurs as a direct result of the TSR2+RFK protein treatment rather than indirectly through inhibition of angiogenesis. Whereas, the TSR2+RFK and TSR2 proteins reduce vessel density to comparable levels, the TSR2+RFK protein has a more profound effect on tumor cell apoptosis. In addition, this protein significantly decreases tumor cell proliferation in vivo.

[0028] The data presented here indicate that inclusion of the additional sequence DKRFK (SEQ ID NO: 2) at the N-terminus of the TSR2 protein to yield the TSR2+RFK protein significantly increases the anti-tumor activity of the protein. The difference in activity of the protein that contains the DKRFK (SEQ ID NO: 2) sequence as compared to the one that does not may be due to the activation of TGFβ, an increased affinity for heparan sulfate proteoglycans or an unidentified activity of this sequence. The additional sequence in the TSR2+RFK protein includes the RFK sequence that reportedly activates TGFβ (Schultz-Cherry, S., et al., J. Biol. Chem., 270: 7304-7310, 1995). In experiments using the culture supernatant from B16F10 cells, it was shown that the TSR2+RFK protein activates TGFβ, while the TSR2 protein does not. TGFβ has pleiotropic effects on tumor growth. At early stages of tumorigenesis, TGFβ may act as a tumor suppressor gene (Engle, S. J., et al., Cancer Research, 59: 3379-3386, 1999; Tang, B., et al., Nature Med., 4: 802-807, 1998). TGFβ can induce apoptosis of several different tumor cell lines (Guo, Y. and Kypianou, N., Cancer Research, 59: 1366-1371, 1999 and references therein). At later stages of tumor growth, TGFβ can stimulate angiogenesis through the recruitment of inflammatory and stromal cells (Pepper, M., Cytokine & Growth Factor Reviews, 8: 21-43, 1997).

[0029] The data presented here indicate that recombinant proteins that include the TSRs have therapeutic value as inhibitors of tumor growth. They act to inhibit angiogenesis and to induce tumor cell apoptosis. Since these mechanisms of action are probably different from other inhibitors of neoplasia, a combinational approach that includes TSRs with other inhibitors of tumor growth can be an effective treatment for cancer.

[0030] The RFK sequence mediates the ability of TSP-1 to activate TGFβ (Schultz-Cherry, S. et al., J. Biol. Chem. 270:7304-7310, 1995). Mutation of the arginine residue (R) to glutamine (Q) abolishes the ability of synthetic peptides to activate TGFβ. A recombinant version of the TSR2+RFK protein was produced in which Q is substituted for R. This protein is designated TSR2+QFK. In the B16F10 experimental tumor model, TSR2+QFK is less effective as an inhibitor of tumor growth than TSR2+RFK (FIG. 8). The activity of TSR2+QFK is comparable to that of TSR2. A recombinant version of all three type 1 repeats of mouse TSP-2 has also been prepared. TSP2 has the sequence RIR in the position where TSP-1 has RFK, and has been reported to be unable to activate TFGβ (Schultz-Cherry, S. et al, J. Biol. Chem. 270:7304-7310, 1995). The recombinant protein that includes all three TSRs of mouse TSP-2 is significantly less potent as an inhibitor of B16F10 tumor growth than all three type 1 repeats of human TSP-1 (FIG. 9).

[0031] The ability of TSR2+RFK to inhibit tumor growth was also evaluated in the presence of a soluble form of the TGFβ receptor. Systemic injection of this reagent has been shown to inhibit endogenous TGFβ activity (Smith, J. D. et al., Circ. Res. 84:1212-22, 1999). Concurrent treatment of B16F10 tumor-bearing mice with TSR2+RFK and the soluble form of the TGFβ receptor resulted in a significant loss of anti-tumor activity (FIG. 8). The level of inhibition of tumor growth under these circumstances was comparable to that observed with the TSR2 or TSR2+QFK proteins. Animals that received control injections of saline with the soluble TGFβ receptor produced tumors that are about 15% larger than those produced in the mice that received only saline. This suggests that TGFβ that is activated by endogenous TSP-1 or by other means is normally suppressing tumor growth.

[0032] Taken together, these data indicate that the ability of the TSR2+RFK protein to inhibit tumor growth is due in part to the activation of TGFβ by the RFK sequence. This conclusion is consistent with the observation that both TSR2+RFK and TGFβ inhibit B16F10 tumor cell proliferation in vitro.

[0033] The recombinant protein is more effective than the intact TSP-1 protein in inhibiting tumor growth. The recombinant protein includes specific active sequences and does not include other domains of TSP-2 or TSP-1 that may decrease the over all activity. See Table 1 for active sequences of TSP-1 (taken from chapter 2, “The Primary Structure of the Thrombospondins” In The Thrombospondin Gene Family (J. C. Adams et al., eds.) Springer-Verlag, Heidelberg (1995. The recombinant protein can be made in large quantities and does not require human tissue as a source of protein. This protein is folded like the wild-type protein and can be more stable in circulation than peptides. It can also be modified to include sequences that will target it to tumor tissue. Because these proteins are derived from portions of human proteins, they should not be immunogenic in humans. TABLE 1 Active Regions of Interest Within Thrombospondin-1 Domain Sequence Function Procollagen NGVQYRN (SEQ ID NO: 4) Anti-angiogenesis homology Type 1 CSVTCG (SEQ ID NO: 5) Cell binding repeats WSXWSXW (SEQ ID NO: 6) Heparin binding GGWSHW (SEQ ID NO: 7) TGF-β and Fibronectin binding RFK TGF-β activation SPWDICSVTCGGGVQKRSR Anti-angiogenesis (SEQ ID NO: 8) Type 2 DVDEC(X)₆C(X)₈CENTDPGYNCLPC Calcium binding repeats (SEQ ID NO: 9)

[0034] In one aspect, the invention comprises polynucleotides or nucleic acid molecules that encode polypeptides whose amino acid sequences are derived from human TSP-1. By the genomic structure, the type 1 repeats of TSP-1 are amino acid residues 359414 (first), amino acid residues 415-473 (second), and 474-531 (third). In another case, the polypeptides encoded by the polynucleotides of the invention are fragments or variants of the immediately aforementioned polypeptides, which have activity that is similar in quality and quantity (for example, plus or minus one order of magnitude in an assay) to the anti-angiogenic and/or apoptosis increasing and/or anti-tumor growth activity of the polypeptides tested in the Examples. A fragment of a TSR2+RFK, TSR2+QFK, or TSR2 polypeptide can, for instance, have up to 30 (any number from 1 to 30) consecutive amino acid residues deleted from the carboxy terminus, relative to SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 21, respectively. The carboxy terminal deletion in a fragment of a polypeptide can be, for example, 5, 10, 15, 20 or 25 consecutive amino acids.

[0035] The polynucleotides that encode the polypeptides described herein can be made by recombinant methods, can be made synthetically, can be replicated by enzymes in in vitro (e.g., PCR) or in vivo systems (e.g., by suitable host cells, when inserted into a vector appropriate for replication within the host cells), or can be made by a combination of methods. The polynucleotides of the invention can include DNA and its RNA counterpart.

[0036] As used herein, “nucleic acid,” “nucleic acid molecule,” “oligonucleotide” and “polynucleotide” include DNA and RNA and chemical derivatives thereof, including phosphorothioate derivatives and RNA and DNA molecules having a radioactive isotope or a chemical adduct such as a fluorophore, chromophore or biotin (which can be referred to as a “label”). The RNA counterpart of a DNA is a polymer of ribonucleotide units, wherein the nucleotide sequence can be depicted as having the base U (uracil) at sites within a molecule where DNA has the base T (thymidine).

[0037] Isolated nucleic acid molecules or polynucleotides can be purified from a natural source or can be made recombinantly. Polynucleotides referred to herein as “isolated” are polynucleotides purified to a state beyond that in which they exist in cells. They include polynucleotides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure polynucleotides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant polynucleotides that have been isolated. The term “isolated” as used herein for nucleic acid molecules, indicates that the molecule in question exists in a physical milieu distinct from that in which it occurs in nature. For example, an isolated polynucleotide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, and may even be purified essentially to homogeneity, for example as determined by agarose or polyacrylamide gel electrophoresis or by A₂₆₀/A₂₈₀ measurements, but may also have further cofactors or molecular stabilizers (for instance, buffers or salts) added.

[0038] The invention further relates to fusion proteins comprising, for example: (1) a polypeptide comprising the three type 1 repeats of human TSP-1, but not comprising the other domains of TSP-1; (2) a polypeptide comprising the second type 1 repeat of human TSP-1, but not comprising the RFK sequence or the other domains of TSP-1; (3) a polypeptide comprising the second type 1 repeat of human TSP-1 and the RFK sequence, but not the other domains of TSP-1; (4) a polypeptide comprising the second type 1 repeat of human TSP-1 and the RFK sequence altered to QFK, but not the other domains of TSP-1; or (5) a polypeptide comprising all three type 1 repeats of human TSP-2, but not the other domains of TSP-2, wherein any one of (1), (2), (3), (4) or (5) is linked to a second moiety not occurring in TSP-1 or TSP-2 as found in nature. The second moiety can be an amino acid, peptide or polypeptide, and can have enzymatic or binding activity of its own. The first moiety can be in an N-terminal location, C-terminal location or internal to the fusion protein. The second moiety can comprise a linker sequence and an affinity ligand, for example.

[0039] Variants of the polypeptide include those having amino acid sequences different from sequences which can be seen as contiguous portions of the sequence in FIG. 7 (which illustrates, for example, amino acid residues 361-530 (SEQ ID NO: 20), 411-473 (SEQ ID NO: 22) and 416-473 (SEQ ID NO: 21)). Variants can have, for instance, several, such as 5 to 10, 1 to 5, or 4, 3, 2 or 1 amino acids substituted, deleted, or added, in any combination, compared to the sequences which are a portion of the sequence in FIG. 7. In one embodiment, variants have silent substitutions, additions and/or deletions that do not significantly alter the properties and activities of the polypeptide. See Table 2 for examples of conservative amino acid substitutions. Variants can also be modified polypeptides in which one or more amino acid residues are modified.

[0040] The invention also encompasses variant polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptides described herein by amino acid sequence. Similarity for a polypeptide is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr and Trp. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., Science 247:1306-1310 (1990). See also Table 2. TABLE 2 Conservative Amino Acid Substitutions Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

[0041] “Sequence identity,” as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., two polynucleotides or two polypeptides. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two polypeptides is occupied by serine, then they are identical at that position. The identity between two sequences is a direct function of the number of matching or identical positions, e.g., if half (e.g., 5 positions in a polymer 10 subunits in length) of the positions in two peptide or compound sequences are identical, then the two sequences are 50% identical; if 90% of the positions, e.g., 9 of 10 are matched, the two sequences share 90% sequence identity. By way of example, the amino acid sequences R₂R₅R₇R₁₀R₆R₃ and R₉R₈R₁R₁₀R₆R₃ have 3 of 6 positions in common, and therefore share 50% sequence identity, while the sequences R₂R₅R₇R₁₀R₆R₃ and R₈R₁R₁₀R₆R₃ have 3 of 5 positions in common, and therefore share 60% sequence identity. The identity between two sequences is a direct function of the number of matching or identical positions. Thus, if a portion of the reference sequence is deleted in a particular peptide, that deleted section is not counted for purposes of calculating sequence identity, e.g., R₂R₅R₇R₁₀R₆R₃ and R₂R₅R₇R₁₀R₃ have 5 out of 6 positions in common, and therefore share 83.3% sequence identity.

[0042] Identity is often measured using sequence analysis software e.g., BLASTN or BLASTP (available at http://www.ncbi.nhn.nih.gov/BLAST/. The default parameters for comparing two sequences (e.g., “Blast”-ing two sequences against each other, http://www.ncbi.nlm.nih.gov/gorf/b12.html) by BLASIN (for nucleotide sequences) are reward for match=1, penalty for mismatch=−2, open gap=5, extension gap=2. When using BLASTP for protein sequences, the default parameters are reward for match=0, penalty for mismatch=0, open gap=11, and extension gap=1.

[0043] When two sequences share “sequence homology,” it is meant that the two sequences differ from each other only by conservative substitutions. For polypeptide sequences, such conservative substitutions consist of substitution of one amino acid residue at a given position in the sequence for another amino acid residue of the same class (e.g., amino acids that share characteristics of hydrophobicity, charge, pK or other conformational or chemical properties, e.g., valine for leucine, arginine for lysine), or by one or more non-conservative amino acid substitutions, deletions, or insertions, located at positions of the sequence that do not alter the conformation or folding of the polypeptide to the extent that the biological activity of the polypeptide is destroyed. Examples of “conservative substitutions” include substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another; or the use of a chemically derivatized residue in place of a non-derivatized residue; provided that the polypeptide displays the requisite biological activity. Two sequences which share sequence homology may called “sequence homologs.”

[0044] Homology, for polypeptides, is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Protein analysis software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0045] Embodiments of the invention include not only the polypeptides consisting of SEQ ID NOs 20, 21, 22, 23, 25 and 27, but also any polypeptide which is at least 70% or 80%, more preferably 90%, and still more preferably, 95% identical to any of the TSP polypeptides identified herein by SEQ ID NO, wherein the polypeptide has at least about the same anti-angiogenic or apoptotic or anti-tumor activity as the polypeptide identified by SEQ ID NO. These activities can be determined by methods described herein, for example, the TUNEL assay for apoptosis (Abe, M. et al., Anal. Biochem. 216:276-84, 1994), the measurement of capillary density for anti-angiogenic effect, as described in Example 2, or the counting of tumor cells to determine cell proliferation or the inhibition thereof. Other methods known in the art can also be used.

[0046] A further embodiment of the invention is a 3TSR, TSR2, TSR2+RFK or TSR2+QFK polypeptide, or an active variant or fragment of any of the foregoing, wherein the region of the polypeptide derived from the human TSP-1 amino acid sequence is flanked on one or both ends by other amino acid residues having an amino acid sequence which is not found as the human TSP-1 amino acid sequence that naturally occurs adjacent to the region. Flanking peptide regions can be, for example, 1, 2, 3, 4 or 5, 5-10, 10-20, or 20-30 amino acid residues in length.

[0047] Further embodiments of the invention are mutants of the 3TSR, TSR2, TSR2+RFK, TSR2+QFK, or 3TSR (TSP2) polypeptides. By “mutant” is meant a polypeptide that includes any change in the amino acid sequence relative to the amino acid sequence of the polypeptide described herein by SEQ ID NO. Such changes can arise either spontaneously or by manipulations by man, by chemical energy (e.g., X-ray), or by other forms of chemical mutagenesis, or by genetic engineering, or as a result of mating or other forms of exchange of genetic information. Mutations include, e.g., base substitutions, deletions, insertions, inversions, translocations, or duplications. Mutant forms of the polypeptides can display either increased or decreased anti-angiogenic activity and either increased or decreased tumor growth inhibition activity relative to the polypeptides identified herein by SEQ ID NO. Such mutants may or may not also comprise additional amino acids derived from the process of cloning, e.g., amino acid residues or amino acid sequences corresponding to full or partial linker sequences.

[0048] Mutants/fragments of the anti-angiogenic and/or anti-tumor polypeptides of the present invention can be generated by PCR cloning. To make such fragments, PCR primers can be designed from known nucleic acid sequences in such a way that each set of primers will amplify a predicted region of the coding sequence for the starting polypeptide. These amplified DNAs are then cloned into an appropriate expression vector, such as the pET22b vector, and the expressed polypeptide can ben tested for its anti-angiogenic activity and anti-tumor activity. Alternatively, mutants and fragments can be produced by chemical synthesis methods.

[0049] Proteins and polypeptides described herein can be assessed for their angiogenic activity by using an assay such as the one described in Tolsma, S. S. et al., J. Cell Biol. 122(2):497-511 (1993), an assay which measures the migration of bovine adrenal capillary endothelial cells in culture, or an assay which tests migration of cells into a sponge containing an agent to be tested for activity. A further test for angiogenesis, which can also be adapted also to test anti-angiogenesis activity, is described in Polverini, P. J. et al., Methods. Enzymol. 198:440-450 (1991). More recently, methods to quantitate inhibition of tumor vascularization, and to quantitate inhibition of corneal neovascularization, were described in Blezinger, P. et al., Nature Biotechnology 17:343-348, 1999.

[0050] Another aspect of the invention relates to a method of producing a polypeptide of the invention, or a variant or fragment thereof, and to expression systems and host cells containing a vector appropriate for expression of a polypeptide of the invention. Cells that express such a polypeptide or a variant or a fragment thereof can be made and maintained in culture, under conditions suitable for expression, to produce protein for isolation. These cells can be procaryotic or eucaryotic. Examples of procayotic cells that can be used for expression (as “host cells”; “cell” including herein cells of tissues, cell cultures, cell strains and cell lines) include Escherichia coli, Bacillus subtilis and other bacteria. Examples of eucaryotic cells that can be used for expression include yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and other lower eucaryotic cells, and cells of higher eucaryotes such as those from insects and mammals. Suitable cells of mammalian origin include primary cells, and cell lines such as CHO, HeLa, 3T3, BHK, COS, 293, and Jurkat cells. Suitable cells of insect origin include primary cells, and cell lines such as SF9 and High five cells. (See, e.g., Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., (containing Supplements up through 2001)).

[0051] In one embodiment, host cells that produce a recombinant polypeptide, variant, or a fragment thereof can be made as follows. A gene encoding a polypeptide variant or fragment described herein can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, virus or other suitable replicon (including vectors suitable for use in gene therapy, such as those derived from adenovirus or others; see, for example Xu, M. et al., Molecular Genetics and Metabolism 63:103-109, 1998) present in the cell as a single copy or multiple copies, or the gene can be integrated in a host cell chromosome. A suitable replicon or integrated gene can contain all or part of the coding sequence for the polypeptide, fragment or variant, operably linked to one or more expression control regions whereby the coding sequence is under the control of transcription signals and linked to appropriate translation signals to permit translation. The vector can be introduced into cells by a method appropriate to the type of host cells (e.g., transformation, electroporation, infection). For expression from the gene, the host cells can be maintained under appropriate conditions (e.g., in the presence of inducer, normal growth conditions, etc.). Proteins or polypeptides thus produced can be recovered (e.g., from the cells, the periplasmic space, culture medium) using suitable techniques.

[0052] The invention also relates to isolated proteins or polypeptides. Isolated polypeptides or fragments or variants can be purified from a natural source or can be made recombinantly. Proteins or polypeptides referred to herein as “isolated” are proteins or polypeptides purified to a state beyond that in which they exist in cells and include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. Thus, the term “isolated” as used herein, indicates that the polypeptide in question exists in a physical milieu distinct from the cell in which its biosynthesis occurs. For example, an isolated polypeptide of the invention can be purified essentially to homogeneity, for example as determined by PAGE or column chromatography (for example, HPLC), but may also have further cofactors or molecular stabilizers added to the purified protein to enhance activity. In one embodiment, proteins or polypeptides are isolated to a state at least about 75% pure; more preferably at least about 85% pure, and still more preferably at least about 95% pure, as determined by Coomassie blue staining of proteins on SDS-polyacrylamide gels.

[0053] “Polypeptide” as used herein indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. This term is also intended to include polypeptides that have been subjected to post-expression modifications such as, for example, glycosylations, acetylations, phosphorylations and the like. In addition, the polypeptides, fragments and variants of the invention can be synthesized, by laboratory or by biosynthetic methods, using amino acids that carry modifications such as glycosylation, acetylation, phosphorylation, or chemical adducts for the purpose of providing a chromophoric, fluorophoric, radioactive, or biotin label, for example.

[0054] Fusion proteins comprising a polypeptide described herein can be produced by a variety of methods. For example, a polypeptide can be produced by the insertion of a TSP gene or portion thereof into a suitable expression vector, such as Bluescript SK +/− (Stratagene), pGEX-4T-2 (Pharmacia), pET-15b, pET-20b(+) or pET-24(+) (Novagen). The resulting construct can be introduced into a suitable host cell for expression. Upon expression, the polypeptide can be purified from a cell lysate by means of a suitable affinity matrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 2, pp. 16.4.1-16.7.8, containing supplements up through 2001).

[0055] Polypeptides of the invention can be recovered and purified from cell cultures by well-known methods. The recombinant polypeptide can be purified by ammonium sulfate precipitation, heparin-Sepharose affinity chromatography, gel filtration chromatography and/or sucrose gradient ultracentrifugation using standard techniques. Further methods that can be used for purification of the polypeptide include ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and high performance liquid chromatography. Known methods for refolding protein can be used to regenerate active conformation if the polypeptide is denatured during isolation or purification.

[0056] Also included in the inventions are compositions containing, as a biological ingredient, a polypeptide of the invention, or a variant, fragment or mutant thereof to inhibit angiogenesis and/or increase apoptosis or inhibit tumor growth by other mechanisms in mammalian tissues, and use of such compositions in the treatment of diseases and conditions characterized by, or associated with, angiogenic activity or misregulated growth. Such methods can involve administration by oral, topical, injection, implantation, sustained release, or other delivery methods that bring one or more of the polypeptides in contact with cells whose growth is to be inhibited.

[0057] The present invention includes a method of treating an angiogenesis-mediated disease or cancer with a therapeutically effective amount of one or more polypeptides having anti-angiogenic activity or anti-tumor activity as described herein. Angiogenesis-mediated diseases can include, but are not limited to, cancers, solid tumors, tumor metastasis, benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas), rheumatoid arthritis, psoriasis, ocular angiogenic diseases (e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis), Osler-Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma, and wound granulation. Polypeptide antiangiogenic agents and/or anti-tumor agents can be used, for example, after surgery or radiation to prevent recurrence of metastases, in combination with conventional chemotherapy, immunotherapy, or various types of gene therapy not necessarily directed against angiogenesis.

[0058] “Cancer” means neoplastic growth, hyperplastic or proliferative growth or a pathological state of abnormal cellular development, and includes solid tumors, nonsolid tumors, and any abnormal cellular proliferation, such as that seen in leukemia. As used herein, “cancer” also means angiogenesis-dependent cancers and tumors, i.e., tumors that require for their growth (expansion in volume and/or mass) an increase in the number and density of the blood vessels supplying them with blood. “Regression” refers to the reduction of tumor mass and size. As used herein, the term “therapeutically effective amount” means the total amount of each active component of the composition or method that is sufficient to show a meaningful benefit to a treated human or other mammal, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. More specifically, for example, a therapeutically effective amount of an anti-angiogenic polypeptide can cause a measurable reduction in the size or numbers of tumors, or in their rate of growth or multiplication, compared to untreated tumors. Other methods of assessing a “therapeutically effective amount,” can include the result that blood vessel formation is measurably reduced in treated tissues compared to untreated tissues.

[0059] One or more anti-tumor polypeptides as described herein may be used in combination with other compositions and procedures for the treatment of diseases. For example, a tumor may be treated conventionally with surgery, radiation, chemotherapy, or immunotherapy, combined with anti-angiogenic polypeptides, and then anti-tumor polypeptides may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize and inhibit the growth of any residual primary tumor.

[0060] The compositions may further contain other agents which either enhance the activity of the protein or complement its activity or use in treatment, such as chemotherapeutic or radioactive agents. Such additional factors and/or agents may be included in the composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Additionally, the compositions of the present invention may be administered concurrently with other therapies, e.g., in conjunction with a chemotherapy, immunotherapy or radiation therapy regimen.

[0061] The anti-tumor composition of the present invention may be a solid, liquid or aerosol and may be administered by any known route of administration. Examples of solid compositions include pills, creams, and implantable dosage units. The pills may be administered orally, the therapeutic creams may be administered topically. The implantable dosage unit may be administered locally, for example at a tumor site, or may be implanted for systemic release of the composition, for example subcutaneously. Examples of liquid compositions include formulations adapted for injection subcutaneously, intravenously, intraarterially, and formulations for topical and intraocular administration. Examples of aerosol formulation include inhaler formulation for administration to the lungs.

[0062] One or more polypeptides described herein can be provided as isolated and substantially purified polypeptides in pharmaceutically acceptable formulations (including aqueous or nonaqueous carriers or solvents) using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes. In general, the combinations can be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route. In addition, the polypeptides can be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor, or implanted so that the polypeptides are slowly released systemically. Osmotic minipumps may also be used to provide controlled delivery of high concentrations of polypeptide through cannulae to the site of interest, such as directly into a growth or into the vascular supply to that growth. The biodegradable polymers and their use are described, for example, in detail in Brem et al. (1991, J. Neurosurg. 74:441-446), which is hereby incorporated by reference in its entirety.

[0063] As used herein, the terms “pharmaceutically acceptable,” as it refers to compositions, carriers, diluents and reagents, represents that the materials are capable of administration to or upon a mammal with a minimum of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified, for example, in liposomes.

[0064] The dosage of the polypeptide of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. It is to be understood that the present invention has application for both human and veterinary use. The methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.

[0065] The present invention also encompasses gene therapy whereby a polynucleotide encoding one or more polypeptides or one or more variants or fragments thereof, is introduced and regulated in a patient. Various methods of transferring or delivering DNA to cells for expression of the gene product protein, otherwise referred to as gene therapy, are disclosed in Gene Transfer into Mammalian Somatic Cells in Vivo, N. Yang (1992) Crit. Rev. Biotechnol. 12(4):335-356, which is hereby incorporated by reference. Gene therapy encompasses incorporation of DNA sequences into somatic cells or germ line cells for use in either ex vivo or in vivo therapy. Gene therapy can function to replace genes, augment normal or abnormal gene function, and to combat infectious diseases and other pathologies.

[0066] Strategies for treating these medical problems with gene therapy include therapeutic strategies such as identifying the defective gene and then adding a functional gene to either replace the function of the defective gene or to augment a slightly functional gene; or prophylactic strategies, such as adding a gene for the product protein that will treat the condition or that will make the tissue or organ more susceptible to a treatment regimen. For example, a gene encoding a polypeptide described herein can be inserted into tumor cells of a patient and thus inhibit angiogenesis.

[0067] Gene transfer methods for gene therapy fall into three broad categories: physical (e.g., electroporation, direct gene transfer and particle bombardment), chemical (e.g., lipid-based carriers, or other non-viral vectors) and biological (e.g., virus-derived vector and receptor uptake). For example, non-viral vectors may be used which include liposomes coated with DNA. Such liposome/DNA complexes may be directly injected intravenously into the patient. It is believed that the liposome/DNA complexes are concentrated in the liver where they deliver the DNA to macrophages and Kupffer cells. These cells are long lived and thus provide long term expression of the delivered DNA. Additionally, vectors or the “naked” DNA of the gene may be directly injected into the desired organ, tissue or tumor for targeted delivery of the therapeutic DNA.

[0068] In vivo gene transfer involves introducing the DNA into the cells of the patient when the cells are within the patient. Methods include using virally mediated gene transfer using a noninfectious virus to deliver the gene in the patient or injecting naked DNA into a site in the patient and the DNA is taken up by a percentage of cells in which the gene product protein is expressed. Additionally, the other methods described herein, such as use of a “gene gun,” may be used for in vitro insertion of anti-angiogenic or anti-tumor polypeptide-encoding DNA and linked regulatory sequences.

[0069] Chemical methods of gene therapy may involve a lipid based compound, not necessarily a liposome, to transfer the DNA across the cell membrane. Lipofectins or cytofectins, lipid-based positive ions that bind to negatively charged DNA, make a complex that can cross the cell membrane and allow the DNA into the interior of the cell. Another chemical method uses receptor-based endocytosis, which involves binding a specific ligand to a cell surface receptor and enveloping and transporting it across the cell membrane. The ligand binds to the DNA and the whole complex is transported into the cell. The ligand gene complex is injected into the bloodstream and then target cells that have the receptor will specifically bind the ligand and transport the ligand-DNA complex into the cell.

[0070] A plasmid or other type of suitable vector can be constructed to facilitate the expression of one or more gene sequences encoding a TSP-derived polypeptide as described herein. Strong promoter regions and enhancer regions may be used to facilitate the construction of such a plasmid. If the plasmid is not to be introduced directly at the site of the tumor cells to be treated, it maybe also be desirable to construct the plasmid such that a segment of DNA encoding a signal peptide is inserted immediately 5′ to the coding region for the polypeptide.

[0071] Many gene therapy methodologies employ viral vectors to insert genes into cells. For example, altered retrovirus vectors have been used in ex vivo methods to introduce genes into peripheral and tumor-infiltrating lymphocytes, hepatocytes, epidermal cells, myocytes, or other somatic cells. These altered cells are then introduced into the patient to provide the gene product from the inserted DNA.

[0072] Viral vectors have also been used to insert genes into cells using in vivo protocols. To direct the tissue-specific expression of foreign genes, cis-acting regulatory elements or promoters that are known to be tissue-specific can be used. Alternatively, this can be achieved using in situ delivery of DNA or viral vectors to specific anatomical sites in vivo. For example, gene transfer to blood vessels in vivo was achieved by implanting in vitro transduced endothelial cells in chosen sites on arterial walls. The virus infected surrounding cells which also expressed the gene product. A viral vector can be delivered directly to the in vivo site, by a catheter for example, thus allowing only certain areas to be infected by the virus, and providing long-term, site specific gene expression. In vivo gene transfer using retrovirus vectors has also been demonstrated in mammary tissue and hepatic tissue by injection of the altered virus into blood vessels leading to the organs.

[0073] Viral vectors that have been used for gene therapy protocols include but are not limited to, retroviruses, other RNA viruses such as poliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV40, vaccinia and other DNA viruses. Replication-defective murine retroviral vectors have been widely utilized gene transfer vectors.

[0074] Carrier mediated gene transfer in vivo can be used to transfect foreign DNA into cells. The carrier-DNA complex can be conveniently introduced into body fluids or the bloodstream and then site-specifically directed to the target organ or tissue in the body. Both liposomes and polycations, such as polylysine, lipofectins or cytofectins, can be used. Liposomes can be developed which are cell specific or organ specific and thus the foreign DNA carried by the liposome will be taken up by target cells. Injection of immunoliposomes that are targeted to a specific receptor on certain cells can be used as a convenient method of inserting the DNA into the cells bearing the receptor. Another carrier system that has been used is the asialoglycoprotein/polylysine conjugate system for carrying DNA to hepatocytes for in vivo gene transfer.

[0075] The gene therapy protocol for transfecting anti-tumor polypeptides into a patient may either be through integration of a gene encoding a polypeptide into the genome of the cells, into minichromosomes or as a separate replicating or non-replicating DNA construct in the cytoplasm or nucleoplasm of the cell. Anti-angiogenic polypeptide expression may continue for a long period of time or the carrier-DNA complex may be reinjected periodically to maintain a desired level of the polypeptide in the cell, the tissue or organ or a determined blood level.

[0076] Gene transfer into muscle cells especially has been tested and found to be safe. In a recent test, biologically active endostatin was produced by expression plasmids injected into muscle cells, and inhibited tumor vascularization and the growth of subcutaneous tumors and metastatic lung tumors. (Blezinger, P. et al., Nature Biotechnology 17:343-348, 1999). An appropriate dose of plasmid can be injected intramuscularly, for example, once weekly.

[0077] Peptide phage display libraries have been used to identify peptides that bind specifically to the vascular endothelial cells of different organs such as lung, skin, pancreas and intestine (Rajotte, D. et al., J. Clin. Invest., 102:430-437, 1998). The individual binding properties of the vasculature of specific organs can be used in targeting the delivery of an agent, such as a polypeptide, to those vascular cells.

[0078] Peptides found to bind to the endothelial cells of capillaries supplying blood to individual organs can be produced in fusion with the TSP-derived polypeptides of the invention, to provide a chemical address to target a particular tissue type of organ. For example, the expression vector described above can be engineered to have a coding sequence for a targeting peptide immediately 5′ or immediately 3′ of the coding sequence for a TSP-derived polypeptide of the invention. Such vectors can be administered to a tumor-bearing mammal, such as a human, at the site of one or more tumors, or at a distant site, to provide a source of fusion polypeptide by expression of the fusion gene on the vector. Alternatively, a recombinant fusion protein of a targeting peptide and a TSP-derived polypeptide can be produced in host cells cultured in a laboratory or production facility, using an expression vector or using host cells otherwise genetically altered to produce the fusion polypeptide.

[0079] Not only have peptides been found that bind specifically to the vascular endothelial cells of different organs, but peptides have been found that bind specifically to molecules exposed on the surface of tumor cells and angiogenic tumor vasculature. See, for example, Burg, M. A. et al., Cancer Research, 59:2869-2874, 1999, in which phage display of a peptide library was used to select for phages binding to NG2 proteoglycan, which is expressed on the surface of several different types of tumors.

EXAMPLES Example 1 Production and Characterization of Recombinant Polypeptides

[0080] Human TSP-1 and Recombinant Proteins.

[0081] Human TSP-1 was purified from the supernatant of thrombin-treated platelets as described previously (Adams, J. C. and Lawler, J, Mol. Bio. Cell 5: 423-437, 1994). Recombinant proteins that included sequences from the type 1 repeats were prepared by PCR using the full-length cDNA for human TSP-1 as a template. A DNA segment encoding a recombinant protein containing all three TSRs of TSP-1 (3TSR, amino acids 361-530; SEQ ID NO: 20) was prepared using the forward primer 475htsp1f (GAT GAT CCC GGG GAC GAC TCT GCG GAC GAT GGC; SEQ ID NO: 10) and the reverse primer 476htsp1r (GAT ACC GGT AAT TGG ACA GTC CTG CTT G; SEQ ID NO: 11). A DNA segment encoding a recombinant protein that contains the second TSR (TSR2, amino acids 416-473; SEQ ID NO: 21) was prepared using the forward primer 537htsp1f (GAT GAT CCC GGG CAG GAT GGT GGC TGG AGC; SEQ ID NO: 12) and the reverse primer 515htsp1r (GAT ACC GGT GAT GGG GCA GGC GTC TTT CTT; SEQ ID NO: 13). To evaluate the role of TGFβ activation on the effect of this recombinant protein, a DNA segment encoding a longer version of the second TSR (TSR2+RFK, amino acids 411-473; SEQ ID NO: 22) that includes the RFK sequence was synthesized using the forward primer 514htsp1f (GAT GAT CCC GGG GAC AAG AGA TTT AAA CAG; SEQ ID NO: 14) and the reverse primer 515htsp1r. All three PCR products were cloned between the XmaI and the AgeI sites of the vector pMT/BiP/V5-HisA (Invitrogen, Carlsbad, Calif.). The recombinant proteins included the vector-derived amino acid sequence RSPWG (SEQ ID NO: 15) at the NH₂-terminus and TGHHHHHH (SEQ ID NO: 16) at the COOH-terminus. The fidelity of the PCR products was verified by nucleotide sequencing. Each expression vector was cotransfected into Drosophila S2 cells with the selection vector pCoHYGRO according to the manufacturer's protocols (Invitrogen). Transfected cells were selected with hygromycin B and the expression of recombinant polypeptides was monitored by western blotting using the polyclonal antibody R3 that was raised against a fusion protein that contained all three TSRs of TSP-1 (Legrand, C. et al., Blood, 79:1995-2003, 1992). For large-scale preparation of recombinant protein, S2 cells were grown in serum-free media for five days. The culture supernatant was centrifuged to remove the cells and dialyzed against 20 mM NaPO₄ (pH 7.8) and 500 mM NaCl. The dialysate was applied to a column of ProBond resin (Invitrogen). The column was eluted with 20 mM NaPO₄ (pH 6.0), 500 mM NaCl, 500 mM imidazole. The protein eluted with 500 mM imidazole was dialyzed against 20 mM NaPO₄ (pH 7.0), and 500 mM NaCl and 1% sucrose was added prior to storage.

[0082] Cell Culture.

[0083] Human dermal microvessel endothelial cells (HDMEC) were isolated by the procedure of Richard et al., Exp. Cell Res. 240:1-6 (1998). The cells were cultured in Vitrogen precoated dishes, and maintained in EBM (Clonetics Corp, San Diego, Calif.) containing 20% fetal bovine serum (FBS), 1 mg/ml hydrocortisone acetate, 5×10⁻⁵ M dibutyryl-cAMP, 200 U/ml penicillin, 100 U/ml streptomycin, 250 mg/ml amphotericin and 2-5 ng/ml VEGF. Murine melanoma B16F10 and murine Lewis lung carcinoma cells were obtained from the ATCC, and were maintained in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% FBS, 50 mg/ml penicillin, 50 U/ml streptomycin, and 2 mM glutamine (supplemented DMEM). Mink lung epithelial cells (MLECs-Clone 32) that have been stably transfected with an 800 bp fragment of the plasminogen activator inhibitor-1 (PAI-1) promoter fused to the firefly luciferase reporter gene were kindly provided by Dr. Daniel Rifkin (Abe, M. et al., Anal. Biochem. 216:276-284, 1994). The transfected MLECs were maintained in supplemented DMEM.

[0084] Assay for TGFβ Activation.

[0085] B16F10 cells (2.5×10⁵) were plated in a T25 flask and grown overnight in supplemented DMEM. The cells were rinsed once with 1.0 ml of serum-free DMEM and 2.5 ml of serum-free DMEM containing 5.0 mg/ml of the TSR, or TSR+RFK recombinant protein were added. After an overnight incubation, conditioned media was collected and centrifuged at 12,000 rpm for 5 min to remove cellular debris. Undiluted media was used to determine the level of active TGFβ. The level of total TGFβ was determined by incubating the media at 80° C. for 10 min and diluting 1:5 in DMEM. One hundred microliters of conditioned media were added to wells of a 96-well plate containing 3×10⁴ MLECs and the plates were incubated overnight. A standard curve was constructed by incubating 7.8 to 250 pg/ml of purified TGFβ with the MLECs® & D Systems, Inc. Minneapolis, Minn.) in DMEM. The media was removed and the cells were washed 3 times with cold PBS prior to the addition of 100 ml of lysis buffer (PharMingen, San Diego, Calif.). Luciferase activity was measured using the Enhanced Luciferase Assay Kit PharMingen).

[0086] In Vitro Migration Assay.

[0087] Cells at passage 7-10 were serum-starved and maintained in EBM with 0.1% BSA (control media) for 20 hours before trypsinization to harvest the cells. Cells were washed in EBM twice and resuspended in control media at a concentration of 1×10⁶ cells/ml. Two hundred ml of cells were packed down and kept frozen. They were lysed and serially diluted by CyQuant reagent (Molecular Probe, Eugene, Oreg.) to be used for standard curve construction.

[0088] Transwell membrane (24-well polycarbonate membrane, 8 mM pore size from Corning Costar Corporation, Cambridge, Mass.) coated with Vitrogen (30 mg/ml, Invitrogen) on both sides was used for chemotactic migration experiments. Coated transwells were inverted, 100 ml of cell suspension applied to the top of the membrane and covered by the bottom plate carefully so that the cell suspension stayed on top of the membrane. The cells were allowed to adhere to the coated membrane for two hours in an incubator at 37° C. with 5% CO₂. After the adhesion incubation, the plates were re-inverted, the bottom wells were filled with 0.4 ml control media and the top wells were filled with 0.1 ml control media containing testing reagents. The plate was returned to the incubator for 3.5 hours for the cells to migrate. At the end of the incubation, the transwells were washed in PBS and the cells on the bottom side of the membrane (unmigrated cells) were wiped away with a cotton swab. The membranes were cut out by scalpel and placed into 96-well plates and frozen at −80° C. overnight. Two hundred ml of CyQuant reagent were added to each well. Fluorescence reading was done 16 hours later with a SpectraFluor plate reader with excitation at 485 nm and emission at 535 μm. The number of cells migrated was then calculated based on the standard curve.

[0089] Characterization of Recombinant Proteins

[0090] Three recombinant forms of the type 1 repeats of human TSP-1 have been produced in S2 cells. A particular example of a polypeptide comprising the three type 1 repeats of human TSP-1, but not comprising the other domains of TSP-1 designated “3TSR,” has been made, and contains amino acids 361-530 of human TSP-1. This protein has a predicted molecular weight of 20,520 Daltons and an apparent molecular weight of 25,000 Daltons on SDS-PAGE, suggesting that carbohydrates are added by post-translational modification. The 3TSR, TSR+RFK and TSR recombinant proteins were electrophoresed on a 4%-15% gradient gel. The gel was cut in half and duplicate lanes were stained with Coomassie blue, or western blotted with a polyclonal antibody to all three TSRs of human TSP-1.

[0091] Each TSR contains six cysteine residues. Consistent with the presence of intrachain disulfide bonds in this protein, the 3TSR protein migrates more rapidly in the absence of reducing agent during SDS-PAGE (Panetti, T. S. et al., J. Biol. Chem., 274:430-437, 1999). A second construct, designated TSR2+RFK, contains the second type 1 repeat of human TSP-1 (amino acids 411-473; SEQ ID NO: 22) and includes the DKRFK (SEQ ID NO: 2) sequence in the NH₂-terminal region. The RFK sequence has been shown to be necessary and sufficient for activation of TGFβ (Schultz-Cherry, S. et al., J. Biol. Chem., 270:7304-7310, 1995). An equivalent region of TSP-1 (amino acids 416-473; SEQ ID NO: 21) that excludes the DKRFK (SEQ ID NO: 2) sequence, designated TSR2, has also been expressed. The average level of protein expression is comparable for all three proteins (approximately 24 μg of purified protein from 1 ml of conditioned media). All three proteins react with a polyclonal antibody, designated R3, that was raised against a bacterial fusion protein composed of the type 1 repeats fused to β-galactosidase (Legrand, C. et al., Blood, 79:1995-2003, 1992). Taken together, the data indicate that the recombinant proteins produced in S2 cells are similar to the type 1 repeats in the native protein.

[0092] Functionally, the recombinant TSR-containing proteins are similar to the native TSRs in that they activate TGFβ and inhibit endothelial cell migration. In media conditioned by B16F10 melanoma cells, 6.3% of the total TGFβ is in the active form (FIG. 1). Addition of 5 μg/ml of the TSR2+RFK protein to conditioned media increases the level of the active form to 30% of the total TGFβ. By contrast, addition of TSR2 does not result in an increase in the level of active TGFβ in the conditioned media (4.8%). As shown in FIGS. 2A and 2B, TSP-1 is a potent inhibitor of endothelial cell migration in vitro. This inhibition is dose-dependent up to 100 ng/ml of TSP-1, but concentrations of TSP-1 above this level are less effective. This biphasic response has been reported by others (Tolsma, S. S. et al., J. Cell Biol., 122: 497-511, 1993). The 3TSR, TSR2 and TSR2+RFK recombinant proteins also inhibit endothelial migration with responses that are similar to TSP-1, on a weight basis. All three recombinant proteins are maximally effective in inhibiting endothelial cell migration at 10 ng/ml (FIGS. 2A and 2B).

Example 2 Testing Effect of Recombinant Polypeptides on Tumor Growth

[0093] Primary Tumor Growth Assay.

[0094] The proteins for injection were mixed with Polymyxin B-Agarose (Sigma) for 30 minutes at room temperature to remove endotoxin. The endotoxin levels were less than 0.05 EU/mg as determined using the QCL-1000 assay kit (Biowhittaker, Walkersville, Md.). Proteins were filter-sterilized and the protein concentration was determined prior to injection.

[0095] Five to eight week old C57BL/6 mice (Taconic, Germantown, N.Y.) were acclimated, caged in groups of four or less and their backs were shaved. Cultured B16F10 melanoma or Lewis lung carcinoma cells (1×10⁶) were inoculated subcutaneously on the back of each mouse. Tumors were measured with a dial-caliper and the volumes were determined using the formula width²×length×0.52. The treatment began 4 days after inoculation of the tumor cells. The therapeutic groups received TSP-1 or recombinant TSP-1 proteins IP daily, and the negative control group received comparable injections of saline alone. The experiments were terminated and mice sacrificed when the control mice began to die, usually at day 16 post-tumor cell injection.

[0096] Histological Examination.

[0097] The tumor tissues were cut, fixed with neutral buffered formaldehyde, and embedded in paraffin according to standard histological procedures. Hematoxylin and eosin staining was employed for tissue morphology examination. Blood vessels were immunochemically stained by anti-CD31 antibody with a Vectastain ABC kit (Vector Laboratories, Burlingame, Calif.). Tumor cell apoptosis was detected by TUNEL assay (Abe, M. et al., Anal. Biochem, 216:276-84, 1994). The number of blood vessels was recorded by counting ten high-power fields. Tumor cell proliferation and the apoptotic index were estimated by the percentage of cells scored under a light microscope. A minimum of 1,000 cells was counted in each tumor sample.

[0098] Inhibition of Experimental Tumor Growth by Recombinant Proteins

[0099] The B16F10 and the Lewis lung carcinoma models of experimental tumor growth have been used to assay the effect of the recombinant proteins. These models have been used extensively to assay the activity of the anti-angiogenic proteins endostatin, angiostatin and anti-thrombin III (O'Reilly, M. S. et al. Science, 285:1926-8, 1999; O'Reilly, M. S. et al, Cell 88:277-85, 1997; Boehm, T. et al., Nature 390:404407, 1997). Systemic injection of TSP-1 (0.25 mg/ml) into tumor-bearing mice inhibits tumor growth (FIG. 3A) by 56% on the twelfth treatment day. At higher doses less inhibition is observed and a dose of 2.5 mg/kg/day has no effect on tumor growth. The 3TSR protein is a less effective inhibitor of tumor growth at 0.25 mg/kg/day than the intact protein (FIG. 3B). By contrast, at dosages of 1.0 mg/kg/day and greater, the 3TSR protein is more effective than TSP-1. Tumor volume is reduced by 81% by the 3TSR protein with a dose of 2.5 mg/kg/day and it remains maximally active at 10 mg/kg/day.

[0100] The inhibitory effect of the 3TSR protein is also observed in mice bearing experimental tumors that are formed by subcutaneous injection of Lewis lung carcinoma cells. At 2.5 mg/kg/day, the 3TSR protein inhibits tumor growth by 73% on treatment day 12 (FIG. 4). The TSR2+RFK protein is equally as effective as the 3TSR protein on a weight basis. For B16F10 and Lewis lung carcinoma cell-derived tumors, this protein inhibits tumor growth by 86% and 77%, respectively (FIGS. 4 and 5A).

[0101] To explore the potential involvement of the RFK sequence in the inhibition of B16F10 tumor growth, the TSR2+RFK, and TSR2 proteins, have been assayed. The tumor inhibition effect of the TSR2+RFK protein was comparable to that of the 3TSR protein at the various doses used (FIGS. 4B and 6A). By contrast, TSR2 is significantly less effective (FIG. 6B). At 0.25 mg/kg/day, the tumor growth in the mice treated with the TSR2 protein was indistinguishable from the control group. At 1.0 mg/kg/day, the TSR2+RFK protein inhibited tumor growth by 80%, while the TSR2 protein only inhibited growth by 38% on the twelfth day of treatment. At the highest dose tested (2.5 mg/kg/day), the difference was less pronounced with the TSR2+RFK and TSR2 proteins inhibiting tumor growth by 86% and 68%, respectively.

[0102] To better understand the effect of the recombinant protein treatment on tumor growth, we have determined the rate of proliferation, the apoptotic index and the capillary density for tumors in the saline control group, mice treated with the TSR2+RFK protein or mice treated with the TSR2 protein at an intermediate dose (1.0 mg/kg/day) where the largest effect of inclusion of the RFK sequence is observed. Tumors were removed and fixed on the twelfth treatment day. Capillaries were visualized with anti-CD31 antibody and apoptotic cells were identified by TUNEL. Tumors displayed a 71% and 64% reduction in capillary density when the mice were treated with the TSR2+RFK and TSR2 proteins, respectively (FIG. 6A). Whereas the tumors from mice that were treated with the TSR2+RFK protein displayed a 4-fold increase in tumor cell apoptosis p<0.005), the tumors from the mice that were treated with the TSR2 protein displayed only a 1.9-fold increase in apoptosis (p≦0.025) (FIG. 6B). The TSR2+RFK protein also reduced the percentage of PCNA positive tumor cells. The number of proliferating cells was decreased by 35% by treatment with the TSR2+RFK protein (p<0.005), while treatment with the TSR2 protein reduced tumor cell proliferation by 7.8% (p<0.05) (FIG. 6C).

[0103] Constructs for Expression of TSR2+QFK and 3TSR (TSP-2) Polypeptides

[0104] A recombinant protein in which arginine (413) was mutated to glutamine was prepared using the forward primer 605 htsp1f (GAT GAT CCC GGG GAC AAG CAA TTT AAA CAG GAT GG; SEQ ID NO: 17) and the reverse primer 515 htsp1r (GAT ACC GGT GAT GGG GCA GGC GTC TTT CTT; SEQ ID NO: 13). The resulting PCR product contains the glutamine codon CAA instead of the arginine codon AGA. The template for the PCR reaction was human thrombospondin-1 cDNA and the cloning sites were XmaI and AgeI in the pMT/BiP/V5-HisA vector. This approach is equivalent to that used for the construction of TSR2+RFK.

[0105] This approach was also used to construct the recombinant protein with all three TSRs of mouse TSP-2. In this case, a full-length cDNA clone for mouse TSP-2 was used as the template and the primers 544 mtsp2f (GAT GAT CCC GGG GAT GAG GGC TGG TCT CCG; SEQ ID NO: 18) and 545 mtsp2r (GAT ACC GGT AAT AGG GCA GCT TCT CTT; SEQ ID NO: 19) were used. The 3TSR (TSP-2) polypeptide of mouse consists of amino acids Asp 381 through Ile 550 (SEQ ID NO: 25) as shown in FIG. 10 (SEQ ID NO: 24). By similar methods, a 3TSR (TSP2) polypeptide of human could be made. See, for example, National Center for Biotechnology Information Accession No. NM 003247, Homo sapiens thrombospondin 2 mRNA. A polypeptide produced by these methods would consist of amino acids Glu 381 through Val 550.

[0106] Inhibition of B16F10 Experimental Tumor Growth by Various Recombinant Proteins TSR2, TSR2+RFK and TSR2+QFK.

[0107] Each treatment group contained six five- to eight-week old C57BL/6 mice. Treatment was initiated four days after subcutaneous injection of 1×10⁶ B16F10 melanoma cells. The mice received one IP injection each day of saline, TSR2, TSR2+RFK or TSR2+QFK (SEQ ID NO: 23). A soluble form of the TGFβ receptor (100 μg/mouse/injection) (Smith, J. D. et al., Circulation Research 84:1212-1222, 1999) was included in the saline or TSR2+RFK injections on treatment day 1 and 7 for two additional experimental groups. The recombinant TSR-containing proteins were used at 1.0 mg/kg/day. Results are shown in FIG. 8.

[0108] Inhibition of B16F10 Experimental Tumor Growth by All Three Type 1 Repeats of Human TSP-1 or Mouse TSP-2.

[0109] Each treatment group contained six five- to eight-week old C57BL/6 mice. Treatment was initiated four days after subcutaneous injections of 1×10⁶ B16F10 melanoma cells. The mice received one IP injection each day, of saline, all three type 1 repeats of human TSP-1 [3TSR (TSP1) in FIG. 9] or all three type 1 repeats of mouse TSP-2 [3TSR (TSP2) in FIG. 9]. Recombinant proteins were used at a dose of 2.5 mg/kg/day.

[0110] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

1 27 1 1150 PRT Homo sapiens 1 Asn Arg Ile Pro Glu Ser Gly Gly Asp Asn Ser Val Phe Asp Ile Phe 1 5 10 15 Glu Leu Thr Gly Ala Ala Arg Lys Gly Ser Gly Arg Arg Leu Val Lys 20 25 30 Gly Pro Asp Pro Ser Ser Pro Ala Phe Arg Ile Glu Asp Ala Asn Leu 35 40 45 Ile Pro Pro Val Pro Asp Asp Lys Phe Gln Asp Leu Val Asp Ala Val 50 55 60 Arg Thr Glu Lys Gly Phe Leu Leu Leu Ala Ser Leu Arg Gln Met Lys 65 70 75 80 Lys Thr Arg Gly Thr Leu Leu Ala Leu Glu Arg Lys Asp His Ser Gly 85 90 95 Gln Val Phe Ser Val Val Ser Asn Gly Lys Ala Gly Thr Leu Asp Leu 100 105 110 Ser Leu Thr Val Gln Gly Lys Gln His Val Val Ser Val Glu Glu Ala 115 120 125 Leu Leu Ala Thr Gly Gln Trp Lys Ser Ile Thr Leu Phe Val Gln Glu 130 135 140 Asp Arg Ala Gln Leu Tyr Ile Asp Cys Glu Lys Met Glu Asn Ala Glu 145 150 155 160 Leu Asp Val Pro Ile Gln Ser Val Phe Thr Arg Asp Leu Ala Ser Ile 165 170 175 Ala Arg Leu Arg Ile Ala Lys Gly Gly Val Asn Asp Asn Phe Gln Gly 180 185 190 Val Leu Gln Asn Val Arg Phe Val Phe Gly Thr Thr Pro Glu Asp Ile 195 200 205 Leu Arg Asn Lys Gly Cys Ser Ser Ser Thr Ser Val Leu Leu Thr Leu 210 215 220 Asp Asn Asn Val Val Asn Gly Ser Ser Pro Ala Ile Arg Thr Asn Tyr 225 230 235 240 Ile Gly His Lys Thr Lys Asp Leu Gln Ala Ile Cys Gly Ile Ser Cys 245 250 255 Asp Glu Leu Ser Ser Met Val Leu Glu Leu Arg Gly Leu Arg Thr Ile 260 265 270 Val Thr Thr Leu Gln Asp Ser Ile Arg Lys Val Thr Glu Glu Asn Lys 275 280 285 Glu Leu Ala Asn Glu Leu Arg Arg Pro Pro Leu Cys Tyr His Asn Gly 290 295 300 Val Gln Tyr Arg Asn Asn Glu Glu Trp Thr Val Asp Ser Cys Thr Glu 305 310 315 320 Cys His Cys Gln Asn Ser Val Thr Ile Cys Lys Lys Val Ser Cys Pro 325 330 335 Ile Met Pro Cys Ser Asn Ala Thr Val Pro Asp Gly Glu Cys Cys Pro 340 345 350 Arg Cys Trp Pro Ser Asp Ser Ala Asp Asp Gly Trp Ser Pro Trp Ser 355 360 365 Glu Trp Thr Ser Cys Ser Thr Ser Cys Gly Asn Cys Ile Gln Gln Arg 370 375 380 Gly Arg Ser Cys Asp Ser Leu Asn Asn Arg Cys Glu Gly Ser Ser Val 385 390 395 400 Gln Thr Arg Thr Cys His Ile Gln Glu Cys Asp Lys Arg Phe Lys Gln 405 410 415 Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser Cys Ser Val Thr 420 425 430 Cys Gly Asp Gly Val Ile Thr Arg Ile Arg Leu Cys Asn Ser Pro Ser 435 440 445 Pro Gln Met Asn Gly Lys Pro Cys Glu Gly Glu Ala Arg Glu Thr Lys 450 455 460 Ala Cys Lys Lys Asp Ala Cys Pro Ile Asn Gly Gly Trp Gly Pro Trp 465 470 475 480 Ser Pro Trp Asp Ile Cys Ser Val Thr Cys Gly Gly Gly Val Gln Lys 485 490 495 Arg Ser Arg Leu Cys Asn Asn Pro Thr Pro Gln Phe Gly Gly Lys Asp 500 505 510 Cys Val Gly Asp Val Thr Glu Asn Gln Ile Cys Asn Lys Gln Asp Cys 515 520 525 Pro Ile Asp Gly Cys Leu Ser Asn Pro Cys Phe Ala Gly Val Lys Cys 530 535 540 Thr Ser Tyr Pro Asp Gly Ser Trp Lys Cys Gly Ala Cys Pro Pro Gly 545 550 555 560 Tyr Ser Gly His Gly Ile Gln Cys Thr Asp Val Asp Glu Cys Lys Glu 565 570 575 Val Pro Asp Ala Cys Phe Asn His Asn Gly Glu His Arg Cys Glu Asn 580 585 590 Thr Asp Pro Gly Tyr Asn Cys Leu Pro Cys Pro Pro Arg Phe Thr Gly 595 600 605 Ser Gln Pro Phe Gly Gln Gly Val Glu His Ala Thr Ala Asn Lys Gln 610 615 620 Val Cys Lys Pro Arg Asn Pro Cys Thr Asp Gly Thr His Asp Cys Asn 625 630 635 640 Lys Asn Ala Lys Cys Asn Tyr Leu Gly His Tyr Ser Asp Pro Met Tyr 645 650 655 Arg Cys Glu Cys Lys Pro Gly Tyr Ala Gly Asn Gly Ile Thr Cys Gly 660 665 670 Glu Asp Thr Asp Leu Asp Gly Trp Pro Asn Glu Asn Leu Val Cys Val 675 680 685 Ala Asn Ala Thr Tyr His Cys Lys Lys Asp Asn Cys Pro Asn Leu Pro 690 695 700 Asn Ser Gly Gln Glu Asp Tyr Asp Lys Asp Gly Ile Gly Asp Ala Cys 705 710 715 720 Asp Asp Asp Asp Asp Asn Asp Lys Ile Pro Asp Asp Arg Asp Asn Cys 725 730 735 Pro Phe His Tyr Asn Pro Ala Gln Tyr Asp Tyr Asp Arg Asp Asp Val 740 745 750 Gly Asp Arg Cys Asp Asn Cys Pro Tyr Asn His Asn Pro Asp Gln Ala 755 760 765 Asp Thr Asp Asn Asn Gly Glu Gly Asp Ala Cys Ala Ala Asp Ile Asp 770 775 780 Gly Asp Gly Ile Leu Asn Glu Arg Asp Asn Cys Gln Tyr Val Tyr Asn 785 790 795 800 Val Asp Gln Arg Asp Thr Asp Met Asp Gly Val Gly Asp Gln Cys Asp 805 810 815 Asn Cys Pro Leu Glu His Asn Pro Asp Gln Leu Asp Ser Asp Ser Asp 820 825 830 Arg Ile Gly Asp Thr Cys Asp Asn Asn Gln Asp Ile Asp Glu Asp Gly 835 840 845 His Gln Asn Asn Leu Asp Asn Cys Pro Tyr Val Pro Asn Ala Asn Gln 850 855 860 Ala Asp His Asp Lys Asp Gly Lys Gly Asp Ala Cys Asp His Asp Asp 865 870 875 880 Asp Asn Asp Gly Ile Pro Asp Asp Lys Asp Asn Cys Arg Leu Val Pro 885 890 895 Asn Pro Asp Gln Lys Asp Ser Asp Gly Asp Gly Arg Gly Asp Ala Cys 900 905 910 Lys Asp Asp Phe Asp His Asp Ser Val Pro Asp Ile Asp Asp Ile Cys 915 920 925 Pro Glu Asn Val Asp Ile Ser Glu Thr Asp Phe Arg Arg Phe Gln Met 930 935 940 Ile Pro Leu Pro Lys Gly Thr Ser Gln Asn Asp Pro Asn Trp Val Val 945 950 955 960 Arg His Gln Gly Lys Glu Leu Val Gln Thr Val Asn Cys Asp Pro Gly 965 970 975 Leu Ala Val Gly Tyr Asp Glu Phe Asn Ala Val Asp Phe Ser Gly Thr 980 985 990 Glu Phe Ile Asn Thr Glu Arg Asp Asp Asp Tyr Ala Gly Phe Val Phe 995 1000 1005 Gly Tyr Gln Ser Ser Ser Arg Phe Tyr Val Val Met Trp Lys Gln Val 1010 1015 1020 Gln Ser Tyr Trp Asp Thr Asn Pro Thr Arg Ala Gln Gly Tyr Ser Gly 1025 1030 1035 1040 Leu Ser Val Lys Val Val Asn Ser Thr Thr Gly Pro Gly Glu His Leu 1045 1050 1055 Arg Asn Ala Leu Trp His Thr Gly Asn Thr Pro Gly Gln Val Arg Thr 1060 1065 1070 Leu Trp His Asp Pro Arg His Ile Gly Trp Lys Asp Phe Thr Ala Tyr 1075 1080 1085 Arg Trp Arg Leu Ser His Arg Pro Lys Thr Gly Phe Ile Arg Val Asn 1090 1095 1100 Met Tyr Glu Gly Lys Lys Ile Met Ala Asp Ser Gly Pro Ile Tyr Asp 1105 1110 1115 1120 Lys Thr Tyr Ala Gly Gly Arg Leu Gly Leu Phe Val Phe Ser Gln Glu 1125 1130 1135 Met Val Phe Phe Ser Asp Leu Lys Tyr Glu Cys Arg Asp Pro 1140 1145 1150 2 5 PRT Homo sapiens 2 Asp Lys Arg Phe Lys 1 5 3 4 PRT Homo sapiens 3 Lys Arg Phe Lys 1 4 7 PRT Homo sapiens 4 Asn Gly Val Gln Tyr Arg Asn 1 5 5 6 PRT Homo sapiens 5 Cys Ser Val Thr Cys Gly 1 5 6 7 PRT Homo sapiens VARIANT 3, 6 Xaa = Any Amino Acid 6 Trp Ser Xaa Trp Ser Xaa Trp 1 5 7 6 PRT Homo sapiens 7 Gly Gly Trp Ser His Trp 1 5 8 19 PRT Homo sapiens 8 Ser Pro Trp Asp Ile Cys Ser Val Thr Cys Gly Gly Gly Val Gln Lys 1 5 10 15 Arg Ser Arg 9 33 PRT Homo sapiens VARIANT 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20 Xaa = Any Amino Acid 9 Asp Val Asp Glu Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys Glu Asn Thr Asp Pro Gly Tyr Asn Cys Leu Pro 20 25 30 Cys 10 33 DNA Artificial Sequence PCR primer 10 gatgatcccg gggacgactc tgcggacgat ggc 33 11 28 DNA Artificial Sequence PCR primer 11 gataccggta attggacagt cctgcttg 28 12 30 DNA Artificial Sequence PCR primer 12 gatgatcccg ggcaggatgg tggctggagc 30 13 30 DNA Artificial Sequence PCR primer 13 gataccggtg atggggcagg cgtctttctt 30 14 30 DNA Artificial Sequence PCR primer 14 gatgatcccg gggacaagag atttaaacag 30 15 5 PRT Artificial Sequence vector-derived amino acid sequence 15 Arg Ser Pro Trp Gly 1 5 16 8 PRT Artificial Sequence vector-derived amino acid sequence 16 Thr Gly His His His His His His 1 5 17 35 DNA Artificial Sequence PCR primer 17 gatgatcccg gggacaagca atttaaacag gatgg 35 18 30 DNA Artificial Sequence PCR primer 18 gatgatcccg gggatgaggg ctggtctccg 30 19 27 DNA Artificial Sequence PCR primer 19 gataccggta atagggcagc ttctctt 27 20 170 PRT Homo sapiens 20 Asp Asp Gly Trp Ser Pro Trp Ser Glu Trp Thr Ser Cys Ser Thr Ser 1 5 10 15 Cys Gly Asn Gly Ile Gln Gln Arg Gly Arg Ser Cys Asp Ser Leu Asn 20 25 30 Asn Arg Cys Glu Gly Ser Ser Val Gln Thr Arg Thr Cys His Ile Gln 35 40 45 Glu Cys Asp Lys Arg Phe Lys Gln Asp Gly Gly Trp Ser His Trp Ser 50 55 60 Pro Trp Ser Ser Cys Ser Val Thr Cys Gly Asp Gly Val Ile Thr Arg 65 70 75 80 Ile Arg Leu Cys Asn Ser Pro Ser Pro Gln Met Asn Gly Lys Pro Cys 85 90 95 Glu Gly Glu Ala Arg Glu Thr Lys Ala Cys Lys Lys Asp Ala Cys Pro 100 105 110 Ile Asn Gly Gly Trp Gly Pro Trp Ser Pro Trp Asp Ile Cys Ser Val 115 120 125 Thr Cys Gly Gly Gly Val Gln Lys Arg Ser Arg Leu Cys Asn Asn Pro 130 135 140 Thr Pro Gln Phe Gly Gly Lys Asp Cys Val Gly Asp Val Thr Glu Asn 145 150 155 160 Gln Ile Cys Asn Lys Gln Asp Cys Pro Ile 165 170 21 58 PRT Homo sapiens 21 Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser Cys Ser Val 1 5 10 15 Thr Cys Gly Asp Gly Val Ile Thr Arg Ile Arg Leu Cys Asn Ser Pro 20 25 30 Ser Pro Gln Met Asn Gly Lys Pro Cys Glu Gly Glu Ala Arg Glu Thr 35 40 45 Lys Ala Cys Lys Lys Asp Ala Cys Pro Ile 50 55 22 63 PRT Homo sapiens 22 Asp Lys Arg Phe Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp 1 5 10 15 Ser Ser Cys Ser Val Thr Cys Gly Asp Gly Val Ile Thr Arg Ile Arg 20 25 30 Leu Cys Asn Ser Pro Ser Pro Gln Met Asn Gly Lys Pro Cys Glu Gly 35 40 45 Glu Ala Arg Glu Thr Lys Ala Cys Lys Lys Asp Ala Cys Pro Ile 50 55 60 23 61 PRT Artificial Sequence engineered polypeptide 23 Gln Phe Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser 1 5 10 15 Cys Ser Val Thr Cys Gly Asp Gly Val Ile Thr Arg Ile Arg Leu Cys 20 25 30 Asn Ser Pro Ser Pro Gln Met Asn Gly Lys Pro Cys Glu Gly Glu Ala 35 40 45 Arg Glu Thr Lys Ala Cys Lys Lys Asp Ala Cys Pro Ile 50 55 60 24 1172 PRT Mus musculus 24 Met Leu Trp Ala Leu Ala Leu Leu Ala Leu Gly Ile Gly Pro Arg Ala 1 5 10 15 Ser Ala Gly Asp His Val Lys Asp Thr Ser Phe Asp Leu Phe Ser Ile 20 25 30 Ser Asn Ile Asn Arg Lys Thr Ile Gly Ala Lys Gln Phe Arg Gly Pro 35 40 45 Asp Pro Gly Val Pro Ala Tyr Arg Phe Val Arg Phe Asp Tyr Ile Pro 50 55 60 Pro Val Asn Thr Asp Asp Leu Asn Arg Ile Val Lys Leu Ala Arg Arg 65 70 75 80 Lys Glu Gly Phe Phe Leu Thr Ala Gln Leu Lys Gln Asp Arg Lys Ser 85 90 95 Arg Gly Thr Leu Leu Val Leu Glu Gly Pro Gly Thr Ser Gln Arg Gln 100 105 110 Phe Glu Ile Val Ser Asn Gly Pro Gly Asp Thr Leu Asp Leu Asn Tyr 115 120 125 Trp Val Glu Gly Asn Gln His Thr Asn Phe Leu Glu Asp Val Gly Leu 130 135 140 Ala Asp Ser Gln Trp Lys Asn Val Thr Val Gln Val Ala Ser Asp Thr 145 150 155 160 Tyr Ser Leu Tyr Val Gly Cys Asp Leu Ile Asp Ser Val Thr Leu Glu 165 170 175 Glu Pro Phe Tyr Glu Gln Leu Glu Val Asp Arg Ser Arg Met Tyr Val 180 185 190 Ala Lys Gly Ala Ser Arg Glu Ser His Phe Arg Gly Leu Leu Gln Asn 195 200 205 Val His Leu Val Phe Ala Asp Ser Val Glu Asp Ile Leu Ser Lys Lys 210 215 220 Ser Cys Gln His Ser Gln Gly Ala Glu Val Asn Thr Ile Ser Glu His 225 230 235 240 Thr Glu Thr Leu His Leu Ser Pro His Ile Thr Thr Asp Leu Val Val 245 250 255 Gln Gly Val Glu Lys Ala Gln Glu Val Cys Thr His Ser Cys Glu Glu 260 265 270 Leu Ser Asn Met Met Asn Glu Leu Ser Gly Leu His Val Met Val Asn 275 280 285 Gln Leu Ser Lys Asn Leu Glu Arg Val Ser Ser Asp Asn Gln Phe Leu 290 295 300 Leu Glu Leu Ile Gly Gly Pro Leu Lys Thr Arg Asn Met Ser Ala Cys 305 310 315 320 Val Gln Glu Gly Arg Ile Phe Ala Glu Asn Glu Thr Trp Val Val Asp 325 330 335 Ser Cys Thr Thr Cys Thr Cys Lys Lys Phe Lys Thr Val Cys His Gln 340 345 350 Ile Thr Cys Ser Pro Ala Thr Cys Ala Asn Pro Ser Phe Val Glu Gly 355 360 365 Glu Cys Cys Pro Ser Cys Ser His Ser Ala Asp Ser Asp Glu Gly Trp 370 375 380 Ser Pro Trp Ala Glu Trp Thr Glu Cys Ser Val Thr Cys Gly Ser Gly 385 390 395 400 Thr Gln Gln Arg Gly Arg Ser Cys Asp Val Thr Ser Asn Thr Cys Leu 405 410 415 Gly Pro Ser Ile Gln Thr Arg Thr Cys Ser Leu Gly Lys Cys Asp Thr 420 425 430 Arg Ile Arg Gln Asn Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser 435 440 445 Cys Ser Val Thr Cys Gly Val Gly Asn Val Thr Arg Ile Arg Leu Cys 450 455 460 Asn Ser Pro Val Pro Gln Met Gly Gly Lys Asn Cys Lys Gly Ser Gly 465 470 475 480 Arg Glu Thr Lys Pro Cys Gln Arg Asp Pro Cys Pro Ile Asp Gly Arg 485 490 495 Trp Ser Pro Trp Ser Pro Trp Ser Ala Cys Thr Val Thr Cys Ala Gly 500 505 510 Gly Ile Arg Glu Arg Ser Arg Val Cys Asn Ser Pro Glu Pro Gln Tyr 515 520 525 Gly Gly Lys Asp Cys Val Gly Asp Val Thr Glu His Gln Met Cys Asn 530 535 540 Lys Arg Ser Cys Pro Ile Asp Gly Cys Leu Ser Asn Pro Cys Phe Pro 545 550 555 560 Gly Ala Lys Cys Asn Ser Phe Pro Asp Gly Ser Trp Ser Cys Gly Ser 565 570 575 Cys Pro Val Gly Phe Leu Gly Asn Gly Thr His Cys Glu Asp Leu Asp 580 585 590 Glu Cys Ala Val Val Thr Asp Ile Cys Phe Ser Thr Asn Lys Ala Pro 595 600 605 Arg Cys Val Asn Thr Asn Pro Gly Phe His Cys Leu Pro Cys Pro Pro 610 615 620 Arg Tyr Lys Gly Asn Gln Pro Phe Gly Val Gly Leu Glu Asp Ala Arg 625 630 635 640 Thr Glu Lys Gln Val Cys Glu Pro Glu Asn Pro Cys Lys Asp Lys Thr 645 650 655 His Ser Cys His Lys Asn Ala Glu Cys Ile Tyr Leu Gly His Phe Ser 660 665 670 Asp Pro Met Tyr Lys Cys Glu Cys Gln Ile Gly Tyr Ala Gly Asp Gly 675 680 685 Leu Ile Cys Gly Glu Asp Ser Asp Leu Asp Gly Trp Pro Asn Asn Asn 690 695 700 Leu Val Cys Ala Thr Asn Ala Thr Tyr His Cys Ile Lys Asp Asn Cys 705 710 715 720 Pro Lys Leu Pro Asn Ser Gly Gln Glu Asp Phe Asp Lys Asp Gly Ile 725 730 735 Gly Asp Ala Cys Asp Glu Asp Asp Asp Asn Asp Gly Val Ser Asp Glu 740 745 750 Lys Asp Asn Cys Gln Leu Leu Phe Asn Pro Arg Gln Leu Asp Tyr Asp 755 760 765 Lys Asp Glu Val Gly Asp Arg Cys Asp Asn Cys Pro Tyr Val His Asn 770 775 780 Pro Ala Gln Ile Asp Thr Asp Asn Asn Gly Glu Gly Asp Ala Cys Ser 785 790 795 800 Val Asp Ile Asp Gly Asp Asp Val Phe Asn Glu Arg Asp Asn Cys Pro 805 810 815 Tyr Val Tyr Asn Thr Asp Gln Arg Asp Thr Asp Gly Asp Gly Val Gly 820 825 830 Asp His Cys Asp Asn Cys Pro Leu Met His Asn Pro Asp Gln Ile Asp 835 840 845 Gln Asp Asn Asp Leu Val Gly Asp Gln Cys Asp Asn Asn Glu Asp Ile 850 855 860 Asp Asp Asp Gly His Gln Asn Asn Gln Asp Asn Cys Pro Tyr Ile Ser 865 870 875 880 Asn Ser Asn Gln Ala Asp His Asp Asn Asp Gly Lys Gly Asp Ala Cys 885 890 895 Asp Ser Asp Asp Asp Asn Asp Gly Val Pro Asp Asp Arg Asp Asn Cys 900 905 910 Arg Leu Val Phe Asn Pro Asp Gln Glu Asp Ser Asp Gly Asp Gly Arg 915 920 925 Gly Asp Ile Cys Lys Asp Asp Phe Asp Asn Asp Asn Val Pro Asp Ile 930 935 940 Asp Asp Val Cys Pro Glu Asn Asn Ala Ile Thr Glu Thr Asp Phe Arg 945 950 955 960 Asn Phe Gln Met Val Pro Leu Asp Pro Lys Gly Thr Thr Gln Ile Asp 965 970 975 Pro Asn Trp Val Ile Arg His Gln Gly Lys Glu Leu Val Gln Thr Ala 980 985 990 Asn Ser Asp Pro Gly Ile Ala Val Gly Phe Asp Glu Phe Gly Ser Val 995 1000 1005 Asp Phe Ser Gly Thr Phe Tyr Val Asn Thr Asp Arg Asp Asp Asp Tyr 1010 1015 1020 Ala Gly Phe Val Phe Gly Tyr Gln Ser Ser Ser Arg Phe Tyr Val Val 1025 1030 1035 1040 Met Trp Lys Gln Val Thr Gln Thr Tyr Trp Glu Asp Lys Pro Ser Arg 1045 1050 1055 Ala Tyr Gly Tyr Ser Gly Val Ser Leu Lys Val Val Asn Ser Thr Thr 1060 1065 1070 Gly Thr Gly Glu His Leu Arg Asn Ala Leu Trp His Thr Gly Asn Thr 1075 1080 1085 Glu Gly Gln Val Arg Thr Leu Trp His Asp Pro Lys Asn Ile Gly Trp 1090 1095 1100 Lys Asp Tyr Thr Ala Tyr Arg Trp His Leu Ile His Arg Pro Lys Thr 1105 1110 1115 1120 Gly Tyr Met Arg Val Leu Val His Glu Gly Lys Gln Val Met Ala Asp 1125 1130 1135 Ser Gly Pro Ile Tyr Asp Gln Thr Tyr Ala Gly Gly Arg Leu Gly Leu 1140 1145 1150 Phe Val Phe Ser Gln Glu Met Val Tyr Phe Ser Asp Leu Lys Tyr Glu 1155 1160 1165 Cys Arg Asp Ala 1170 25 170 PRT Mus musculus 25 Asp Glu Gly Trp Ser Pro Trp Ala Glu Trp Thr Glu Cys Ser Val Thr 1 5 10 15 Cys Gly Ser Gly Thr Gln Gln Arg Gly Arg Ser Cys Asp Val Thr Ser 20 25 30 Asn Thr Cys Leu Gly Pro Ser Ile Gln Thr Arg Thr Cys Ser Leu Gly 35 40 45 Lys Cys Asp Thr Arg Ile Arg Gln Asn Gly Gly Trp Ser His Trp Ser 50 55 60 Pro Trp Ser Ser Cys Ser Val Thr Cys Gly Val Gly Asn Val Thr Arg 65 70 75 80 Ile Arg Leu Cys Asn Ser Pro Val Pro Gln Met Gly Gly Lys Asn Cys 85 90 95 Lys Gly Ser Gly Arg Glu Thr Lys Pro Cys Gln Arg Asp Pro Cys Pro 100 105 110 Ile Asp Gly Arg Trp Ser Pro Trp Ser Pro Trp Ser Ala Cys Thr Val 115 120 125 Thr Cys Ala Gly Gly Ile Arg Glu Arg Ser Arg Val Cys Asn Ser Pro 130 135 140 Glu Pro Gln Tyr Gly Gly Lys Asp Cys Val Gly Asp Val Thr Glu His 145 150 155 160 Gln Met Cys Asn Lys Arg Ser Cys Pro Ile 165 170 26 1172 PRT Homo sapiens 26 Met Val Trp Arg Leu Val Leu Leu Ala Leu Trp Val Trp Pro Ser Thr 1 5 10 15 Gln Ala Gly His Gln Asp Lys Asp Thr Thr Phe Asp Leu Phe Ser Ile 20 25 30 Ser Asn Ile Asn Arg Lys Thr Ile Gly Ala Lys Gln Phe Arg Gly Pro 35 40 45 Asp Pro Gly Val Pro Ala Tyr Arg Phe Val Arg Phe Asp Tyr Ile Pro 50 55 60 Pro Val Asn Ala Asp Asp Leu Ser Lys Ile Thr Lys Ile Met Arg Gln 65 70 75 80 Lys Glu Gly Phe Phe Leu Thr Ala Gln Leu Lys Gln Asp Gly Lys Ser 85 90 95 Arg Gly Thr Leu Leu Ala Leu Glu Gly Pro Gly Leu Ser Gln Arg Gln 100 105 110 Phe Glu Ile Val Ser Asn Gly Pro Ala Asp Thr Leu Asp Leu Thr Tyr 115 120 125 Trp Ile Asp Gly Thr Arg His Val Val Ser Leu Glu Asp Val Gly Leu 130 135 140 Ala Asp Ser Gln Trp Lys Asn Val Thr Val Gln Val Ala Gly Glu Thr 145 150 155 160 Tyr Ser Leu His Val Gly Cys Asp Leu Ile Gly Pro Val Ala Leu Asp 165 170 175 Glu Pro Phe Tyr Glu His Leu Gln Ala Glu Lys Ser Arg Met Tyr Val 180 185 190 Ala Lys Gly Ser Ala Arg Glu Ser His Phe Arg Gly Leu Leu Gln Asn 195 200 205 Val His Leu Val Phe Glu Asn Ser Val Glu Asp Ile Leu Ser Lys Lys 210 215 220 Gly Cys Gln Gln Gly Gln Gly Ala Glu Ile Asn Ala Ile Ser Glu Asn 225 230 235 240 Thr Glu Thr Leu Arg Leu Gly Pro His Val Thr Thr Glu Tyr Val Gly 245 250 255 Pro Ser Ser Glu Arg Arg Pro Glu Val Cys Glu Arg Ser Cys Glu Glu 260 265 270 Leu Gly Asn Met Val Gln Glu Leu Ser Gly Leu His Val Leu Val Asn 275 280 285 Gln Leu Ser Glu Asn Leu Lys Arg Val Ser Asn Asp Asn Gln Phe Leu 290 295 300 Trp Glu Leu Ile Gly Gly Pro Pro Lys Thr Arg Asn Met Ser Ala Cys 305 310 315 320 Trp Gln Asp Gly Arg Phe Phe Ala Glu Asn Glu Thr Trp Val Val Asp 325 330 335 Ser Cys Thr Thr Cys Thr Cys Lys Lys Phe Lys Thr Ile Cys His Gln 340 345 350 Ile Thr Cys Pro Pro Ala Thr Cys Ala Ser Pro Ser Phe Val Glu Gly 355 360 365 Glu Cys Cys Pro Ser Cys Leu His Ser Val Asp Gly Glu Glu Gly Trp 370 375 380 Ser Pro Trp Ala Glu Trp Thr Gln Cys Ser Val Thr Cys Gly Ser Gly 385 390 395 400 Thr Gln Gln Arg Gly Arg Ser Cys Asp Val Thr Ser Asn Thr Cys Leu 405 410 415 Gly Pro Ser Ile Gln Thr Arg Ala Cys Ser Leu Ser Lys Cys Asp Thr 420 425 430 Arg Ile Arg Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser 435 440 445 Cys Ser Val Thr Cys Gly Val Gly Asn Ile Thr Arg Ile Arg Leu Cys 450 455 460 Asn Ser Pro Val Pro Gln Met Gly Gly Lys Asn Cys Lys Gly Ser Gly 465 470 475 480 Arg Glu Thr Lys Ala Cys Gln Gly Ala Pro Cys Pro Ile Asp Gly Arg 485 490 495 Trp Ser Pro Trp Ser Pro Trp Ser Ala Cys Thr Val Thr Cys Ala Gly 500 505 510 Gly Ile Arg Glu Arg Thr Arg Val Cys Asn Ser Pro Glu Pro Gln Tyr 515 520 525 Gly Gly Lys Ala Cys Val Gly Asp Val Gln Glu Arg Gln Met Cys Asn 530 535 540 Lys Arg Ser Cys Pro Val Asp Gly Cys Leu Ser Asn Pro Cys Phe Pro 545 550 555 560 Gly Ala Gln Cys Ser Ser Phe Pro Asp Gly Ser Trp Ser Cys Gly Phe 565 570 575 Cys Pro Val Gly Phe Leu Gly Asn Gly Thr His Cys Glu Asp Leu Asp 580 585 590 Glu Cys Ala Leu Val Pro Asp Ile Cys Phe Ser Thr Ser Lys Val Pro 595 600 605 Arg Cys Val Asn Thr Gln Pro Gly Phe His Cys Leu Pro Cys Pro Pro 610 615 620 Arg Tyr Arg Gly Asn Gln Pro Val Gly Val Gly Leu Glu Ala Ala Lys 625 630 635 640 Thr Glu Lys Gln Val Cys Glu Pro Glu Asn Pro Cys Lys Asp Lys Thr 645 650 655 His Asn Cys His Lys His Ala Glu Cys Ile Tyr Leu Gly His Phe Ser 660 665 670 Asp Pro Met Tyr Lys Cys Glu Cys Gln Thr Gly Tyr Ala Gly Asp Gly 675 680 685 Leu Ile Cys Gly Glu Asp Ser Asp Leu Asp Gly Trp Pro Asn Leu Asn 690 695 700 Leu Val Cys Ala Thr Asn Ala Thr Tyr His Cys Ile Lys Asp Asn Cys 705 710 715 720 Pro His Leu Pro Asn Ser Gly Gln Glu Asp Phe Asp Lys Asp Gly Ile 725 730 735 Gly Asp Ala Cys Asp Asp Asp Asp Asp Asn Asp Gly Val Thr Asp Glu 740 745 750 Lys Asp Asn Cys Gln Leu Leu Phe Asn Pro Arg Gln Ala Asp Tyr Asp 755 760 765 Lys Asp Glu Val Gly Asp Arg Cys Asp Asn Cys Pro Tyr Val His Asn 770 775 780 Pro Ala Gln Ile Asp Thr Asp Asn Asn Gly Glu Gly Asp Ala Cys Ser 785 790 795 800 Val Asp Ile Asp Gly Asp Asp Val Phe Asn Glu Arg Asp Asn Cys Pro 805 810 815 Tyr Val Tyr Asn Thr Asp Gln Arg Asp Thr Asp Gly Asp Gly Val Gly 820 825 830 Asp His Cys Asp Asn Cys Pro Leu Val His Asn Pro Asp Gln Thr Asp 835 840 845 Val Asp Asn Asp Leu Val Gly Asp Gln Cys Asp Asn Asn Glu Asp Ile 850 855 860 Asp Asp Asp Gly His Gln Asn Asn Gln Asp Asn Cys Pro Tyr Ile Ser 865 870 875 880 Asn Ala Asn Gln Ala Asp His Asp Arg Asp Gly Gln Gly Asp Ala Cys 885 890 895 Asp Pro Asp Asp Asp Asn Asp Gly Val Pro Asp Asp Arg Asp Asn Cys 900 905 910 Arg Leu Val Phe Asn Pro Asp Gln Glu Asp Leu Asp Gly Asp Gly Arg 915 920 925 Gly Asp Ile Cys Lys Asp Asp Phe Asp Asn Asp Asn Ile Pro Asp Ile 930 935 940 Asp Asp Val Cys Pro Glu Asn Asn Ala Ile Ser Glu Thr Asp Phe Arg 945 950 955 960 Asn Phe Gln Met Val Pro Leu Asp Pro Lys Gly Thr Thr Gln Ile Asp 965 970 975 Pro Asn Trp Val Ile Arg His Gln Gly Lys Glu Leu Val Gln Thr Ala 980 985 990 Asn Ser Asp Pro Gly Ile Ala Val Gly Phe Asp Glu Phe Gly Ser Val 995 1000 1005 Asp Phe Ser Gly Thr Phe Tyr Val Asn Thr Asp Arg Asp Asp Asp Tyr 1010 1015 1020 Ala Gly Phe Val Phe Gly Tyr Gln Ser Ser Ser Arg Phe Tyr Val Val 1025 1030 1035 1040 Met Trp Lys Gln Val Thr Gln Thr Tyr Trp Glu Asp Gln Pro Thr Arg 1045 1050 1055 Ala Tyr Gly Tyr Ser Gly Val Ser Leu Lys Val Val Asn Ser Thr Thr 1060 1065 1070 Gly Thr Gly Glu His Leu Arg Asn Ala Leu Trp His Thr Gly Asn Thr 1075 1080 1085 Pro Gly Gln Val Arg Thr Leu Trp His Asp Pro Arg Asn Ile Gly Trp 1090 1095 1100 Lys Asp Tyr Thr Ala Tyr Arg Trp His Leu Thr His Arg Pro Lys Thr 1105 1110 1115 1120 Gly Tyr Ile Arg Val Leu Val His Glu Gly Lys Gln Val Met Ala Asp 1125 1130 1135 Ser Gly Pro Ile Tyr Asp Gln Thr Tyr Ala Gly Gly Arg Leu Gly Leu 1140 1145 1150 Phe Val Phe Ser Gln Glu Met Val Tyr Phe Ser Asp Leu Lys Tyr Glu 1155 1160 1165 Cys Arg Asp Ile 1170 27 170 PRT Homo sapiens 27 Glu Glu Gly Trp Ser Pro Trp Ala Glu Trp Thr Gln Cys Ser Val Thr 1 5 10 15 Cys Gly Ser Gly Thr Gln Gln Arg Gly Arg Ser Cys Asp Val Thr Ser 20 25 30 Asn Thr Cys Leu Gly Pro Ser Ile Gln Thr Arg Ala Cys Ser Leu Ser 35 40 45 Lys Cys Asp Thr Arg Ile Arg Gln Asp Gly Gly Trp Ser His Trp Ser 50 55 60 Pro Trp Ser Ser Cys Ser Val Thr Cys Gly Val Gly Asn Ile Thr Arg 65 70 75 80 Ile Arg Leu Cys Asn Ser Pro Val Pro Gln Met Gly Gly Lys Asn Cys 85 90 95 Lys Gly Ser Gly Arg Glu Thr Lys Ala Cys Gln Gly Ala Pro Cys Pro 100 105 110 Ile Asp Gly Arg Trp Ser Pro Trp Ser Pro Trp Ser Ala Cys Thr Val 115 120 125 Thr Cys Ala Gly Gly Ile Arg Glu Arg Thr Arg Val Cys Asn Ser Pro 130 135 140 Glu Pro Gln Tyr Gly Gly Lys Ala Cys Val Gly Asp Val Gln Glu Arg 145 150 155 160 Gln Met Cys Asn Lys Arg Ser Cys Pro Val 165 170 

What is claimed is:
 1. A pharmaceutical composition comprising TSR2+RFK (SEQ ID NO: 22), or a conservative variant thereof, and a pharmaceutically acceptable carrier.
 2. A pharmaceutical composition comprising a TSR2+RFK polypeptide, fragment or variant, wherein said fragment or variant has at least about the same apoptotic or anti-angiogenic activity as TSR2+RFK (SEQ ID NO: 22) and wherein said fragment has up to 30 consecutive amino acids deleted from the carboxy terminus of TSR2+RFK (SEQ ID NO: 22) and wherein said variant is at least 70% identical to TSR2+RFK (SEQ ID NO: 22), and a pharmaceutically acceptable carrier.
 3. The pharmaceutical composition of claim 2, wherein said TSR2+RFK polypeptide variant is at least 80% identical to TSR2+RFK (SEQ ID NO: 22).
 4. The pharmaceutical composition of claim 2, wherein said TSR2+RFK polypeptide variant is at least 90% identical to TSR2+RFK (SEQ ID NO: 22).
 5. The pharmaceutical composition of claim 2, wherein said TSR2+RFK polypeptide variant is at least 95% identical to TSR2+RFK (SEQ ID NO: 22).
 6. A polypeptide consisting of amino acids 411-473 of human TSP-1.
 7. A method of killing cancerous cells in a patient, comprising administering to said patient an amount of TSR2+RFK (SEQ ID NO: 22), or a conservative variant thereof, sufficient to induce apoptosis in said cancerous cells.
 8. The method of claim 7, wherein said cancerous cells are in a tumor.
 9. The method of claim 8, wherein said administration of TSR2+RFK (SEQ ID NO: 22), or said conservative variant thereof, is sufficient to inhibit neovascularization in said tumor.
 10. A method for reducing volume or inhibiting growth of a tumor in a patient, comprising administering an amount of TSR2+RFK (SEQ ID NO: 22), or a conservative variant thereof, sufficient to induce apoptosis in the tumor cells of said tumor.
 11. The method of claim 10, wherein said administration of TSR2+RFK (SEQ ID NO: 22), or said conservative variant thereof, to said patient is sufficient to inhibit neovascularization in said tumor.
 12. A method of decreasing proliferation of tumor cells in a patient, comprising administering to said patient an amount of TSR2+RFK (SEQ ID NO: 22), or a conservative variant thereof, sufficient to induce apoptosis in said tumor cells.
 13. The method of claim 12, wherein said administration of TSR2+RFK (SEQ ID NO: 22), or said conservative variant thereof, to said patient is sufficient to inhibit neovascularization in the tumor or tumors containing said tumor cells.
 14. A method of killing cancerous cells in a patient, comprising administering to said patient an amount of a TSR2+RFK (SEQ ID NO: 22) fragment sufficient to induce apoptosis in said cancerous cells, wherein said TSR2+RFK fragment has at least about the same apoptotic and anti-angiogenic activity as TSR2+RFK and has up to 30 consecutive amino acids deleted from the carboxy terminus of TSR2+RFK.
 15. A method of killing cancerous cells in a patient, comprising administering to said patient an amount of a TSR2+RFK variant sufficient to induce apoptosis in said cancerous cells, wherein said TSR2+RFK variant is at least 70% identical to TSR2+RFK (SEQ ID NO: 22) and has at least about the same apoptotic and anti-angiogenic activity as TSR2+RFK.
 16. The method of claim 15, wherein said TSR2+RFK variant is at least 80% identical to TSR2+RFK (SEQ ID NO: 22).
 17. The method of claim 15, wherein said TSR2+RFK variant is at least 90% identical to TSR2+RFK (SEQ ID NO: 22).
 18. The method of claim 15, wherein said TSR2+RFK variant is at least 95% identical to TSR2+RFK (SEQ ID NO: 22).
 19. A polypeptide comprising the three type 1 repeats of human TSP-1, but not comprising the other domains of TSP-1.
 20. A pharmaceutical composition comprising SEQ ID NO: 20, or a conservative variant thereof, and a pharmaceutically acceptable carrier.
 21. A polypeptide comprising the second type 1 repeat of human TSP-1, and the RFK sequence altered to QFK, but not the other domains of TSP-1.
 22. A composition comprising the polypeptide of claim 21 in a pharmaceutically acceptable carrier.
 23. A polypeptide comprising the second type 1 repeat of human TSP-1 and the RFK sequence, but not the other domains of TSP-1.
 24. A fragment or variant of the polypeptide of claim 23 which has anti-angiogenic activity or anti-tumor growth activity, or both.
 25. A polypeptide comprising all three type 1 repeats of human TSP-2, but not the other domains of TSP-2.
 26. A pharmaceutical composition comprising a human 3TSR (TSP2) polypeptide (SEQ ID NO: 27), or a conservative variant thereof, and a pharmaceutically acceptable carrier. 